Unnatural Amino Acids: Strategies, Designs and Applications in Medicinal Chemistry and Drug Discovery

Abstract

Peptides offer therapeutic agents that sit within a privileged space between small moelcules and larger biologics. Despite examples of their potential to regulate receptors and modulate disease pathways, the development of peptides with drug-like properties remains a challenge. In the quest to optimize physicochemical parameters and improve target selectivity, unnatural amino acids (UAAs) have emerged as critical tools in peptide- and peptidomimetic-based drugs. The utility of UAAs is illustrated by clinically approved drugs such as methyldopa, baclofen, and gabapentin in addition to small drug molecules for example, bortezomib and sitagliptin. In this perspective, we outline the strategy and deployment of UAAs in FDA-approved drugs and their targets. We further describe the modulation of physicochemical properties in peptides using UAAs. Finally, we elucidate how these improved pharmacological parameters and the role played by UAAs impact the progress of analogs in pre-clinical stages with an emphasis on the role played by UAAs.

Graphical Abstract

1. Introduction

The role of peptides in drug design, discovery and development is indispensable with >100 peptide-based drugs in clinics and >500 in clinical and pre-clinical phases.¹ The inception of several advanced technologies, such as DNA-encoded libraries (DELs),² phage,³ mRNA⁴ and yeast⁵ display techniques, has had a motivating impact on the peptide-based drug discovery. Additionally, chemistry-driven techniques such as solid-⁶ and solution⁷-phase synthesis, ligation chemistry,⁸ and recombinant synthesis,⁹ along with the combination of synthetic tools of C-H functionalization,¹⁰ metal-catalysis,¹¹ ring-closing metathesis and macrocyclization^{11, 12} enabled researchers to identify novel scaffolds to optimize biological and physicochemical properties of existing peptides. Peptides are ideal scaffolds for drug discovery,^13–16 but their utility is hampered by low cell permeability, in vivo stability, bioavailability, and oral absorption.¹⁷ Various strategies have been explored to address these challenges such as: (i) peptide bond isosteres^18–20 and backbone modifications, such as depsipeptide,²¹ azapeptide,²² endothiopeptide,²³ retro-inverso peptide,²⁴ fluoroalkene,²⁵ amidine,²⁶ triazole,²⁷ oxetane²⁸ and imidazolone;²⁹ (ii) modification of the terminus;^{30, 31} (iii) macrocyclization;³² and (iv) incorporation of unnatural³³ and modified amino acids.³⁴

The term “proteinogenic” amino acid is utilized for the 22 standard amino acids coded by the genetic code in proteins. However, several non-coded amino acids are known in native proteins and peptides. Post-translational modifications generate non-proteinogenic amino acids as secondary metabolites in microbes (bacteria, fungi), algae, plants, and marine sources.^{35, 36} Over the years, researchers have synthetically developed an incredible array of unnatural amino acids (UAAs), using innovative synthetic methodologies.^37–41 The UAAs represent a versatile tool for medicinal chemistry and drug discovery programs to design and develop new therapeutics.^42–44 UAAs offer several advantages in terms of upgraded structural characteristics such as varied functional groups, tailor-made chemical reactivity, ease of chemical modification, and physicochemical characteristics, including enzymatic stability, desirable lipophilicity, hydrophilicity, and ionic charge.^45–47 UAAs have become a part of several Food and Drug Administration (FDA)-approved drugs and are utilized to develop new bioactive molecular entities and compound optimization campaigns.^48–50 Evidently, unnatural amino acids can increase the molecular diversity in peptides as compared to canonical or proteinogenic amino acids thereby improving their drug-likeness. UAAs have drawn significant attention as versatile building blocks that possess unique physicochemical and pharmacological properties for the construction of new peptides and peptidomimetics.^51–53

This perspective presents a database and analysis of FDA-approved drugs containing UAAs and discusses their impact on the modulation of the physicochemical properties of drug candidates. Further, we highlight the medicinal chemistry and drug discovery programs that rely upon unnatural amino acids for the optimization of activity, efficacy, target selectivity, and associated parameters. The term “unnatural amino acid” has been chosen to define chemically modified proteinogenic amino acids, unusual classes of amino acids, naturally occurring non-proteinogenic amino acids, D-amino acids, N-methylated amino acids, or α-, β-, γ-, and δ-amino acids. Specific examples have been presented to highlight the role of UAAs against therapeutic targets in various diseases.

2. Unnatural amino acids in FDA-approved drugs

With the emergence of advanced synthetic techniques and the availability of structurally diverse UAAs, the number of FDA-approved drugs containing UAAs has increased over the past few decades (Figure 1a).⁴⁸ More than 110 drugs have been approved bearing UAAs (see Poster and Table S1 in supporting information). The pharmacokinetic properties of a naturally occurring peptide can be easily manipulated by introducing designer and unnatural amino acids. Peptides are infamously known for their easily degradable nature and metabolic liabilities on oral administration, as exemplified by insulin. However, the utilization of UAAs in developing peptide-based bioactives has navigated the drug discovery process towards orally stable scaffolds.^{54, 55} As of now, around 44% of FDA-approved UAAs containing drugs are administered through the oral route (Figure 1b), while 48% are collectively given via parenteral routes, including intravenous (IV), intramuscular (IM) and subcutaneous (SC). Novel scaffolds with rationally placed UAAs have shown a wide array of biological activities, as evident from the FDA approvals under various diseased categories (Figure 1c). The largest proportion of FDA approvals are for anti-infectives and anticancer agents, followed by cardiovascular and neurological disorders. Table S1 comprehensively explains the UAAs containing drugs approved by the FDA till now, along with indications, targets, route of administration, year of approval and the amino acid residue present in the drug. The structural details of FDA-approved drugs containing UAAs have been elaborated in a poster in the SI and in Figures S2-S8 with highlighted UAAs.

Figure 1.

(a) Decade-wise distribution of UAAs containing US FDA-approved drugs; (b) Comparison between various routes of administration; (c) Distribution of peptides in different disease areas.

A number of UAAs are present in FDA-approved drugs, demonstrating their utility as clinical agents (Table S1). In some cases, the UAA itself can act as a drug, such as vigabatrin (2009), brivaracetam (2016), and fluorodopa (2019). Cyclic amino acids, amino acids with unnatural side chains, synthetically designed amino acids and conformationally constrained amino acids are widespread in approved drugs.⁵⁶ Importantly, synthetic chemistry developments provide access to the large number of unnatural classes of amino acids possessing unique chemical and biological properties that are employed in drug discovery campaigns.

3. Modulation of the physicochemical properties of peptides using UAAs

Peptides are an intrinsic part of the human physiological and regulatory system, making them a promising launching pad for drug discovery and development. Peptides as drug candidates, however, are often disregarded due to several inherent characteristics. Peptides can display short half-lives due to rapid enzymatic degradation and rapid renal clearance, rendering them of insufficient concentration to act at the target tissue or organ.⁵⁷ The presence of hydrophilic residues can reduce permeability across biological membranes, reducing efficacy. The modulation of these intrinsic characteristics of with UAAs can lead to analogs with more drug-likeliness.^{1, 58} These amino acids, when incorporated into the peptides, alter pharmacological profiles and several physicochemical properties such as lipophilicity, hydrogen bond donor or acceptor nature, stability, membrane permeability, bioavailability, bioactivity, distribution and target selectivity.^{34, 59–63} Most of the peptides don’t follow Lipinski’s rule of five for drug-likeness; therefore, optimization of physicochemical properties and drug-likeness in peptides is more challenging and complex than small-molecules. Some representative examples are discussed below to provide insights into the role of unnatural amino acids in modifying physicochemical properties during medicinal campaigns.

3.1. Modulation of the lipophilicity and stability

Lipophilicity is a key physicochemical property for clinical agents and peptides that play a vital role in optimizing the ADME profile of drugs. Lipophilic changes in peptides have a significant impact on stability, solubility, membrane permeability, potency and selectivity, therefore affecting their pharmacokinetics, pharmacodynamics, and toxicological profiles. Interestingly, DPDPE (1, Figure 2), a Met-enkephalin analog, is a cyclic peptide with D-penicillamine (D-Pen) at positions 2 and 5, linked via a disulfide bridge. Replacement of Tyr¹ with 2’,6’-dimethyltyrosine (Dmt) increased the lipophilicity of the resulting peptide 2 and enhanced analgesic activity at δ- opioid receptors (10-fold) and µ-opioid receptors (35-fold). The presence of methyl groups decreased the peptide hydrogen bonding capability, which boosted diffusion via enhancing lipophilicity.^{64, 65}

Figure 2.

Structures and improved profile of met-enkephalin analogs.

A classic example of increased proteolytic stability is octreotide (4), a synthetic analog of somatostatin (3, Figure 3). Somatostatin is rapidly degraded by enzymes in a human plasma, with a half-life of 1-2 min. Octreotide is a truncated analog of somatostatin, where L-Trp⁸ has been replaced with D-Trp⁸, among other changes. The modifications increased the half-life to 1.5 h in human plasma with similar pharmacological effects. Octreotide can be administered intravenously and subcutaneously and possesses similar bioavailability via both routes.^{66, 67}

Figure 3.

Structures of somatostatin and octreotide.

The potency and metabolic stability of the naturally occurring antimicrobial peptide feleucin-K3 (5, Figure 4) were enhanced by substituting Leu⁴ with α-(4-pentenyl)-Ala. The modified analog feleucin-K59 (6) exhibited improved in vitro and in vivo antimicrobial activities against both standard and drug-resistant strains. The stability study revealed that about 21% of 5 was active after 8 h, which was completely degraded within 24 h of incubation. However, increased stability was observed in 6, and more than 30% of active peptide was detected in plasma after 24 h of incubation.⁶⁸

Figure 4.

Comparative structure of Feleucin-K3 and the derivative Feleucin-K59.

Another example that explains the role of UAAs in improving potency and systemic stability is PHSCN (7, Figure 5). PHSCN prevents invasion and metastasis in the preclinical adenocarcinoma model by activating α5β1 integrin. The replacement of l-histidine and l-cysteine residues with D-histidine and D-cysteine gave a mixed-chirality peptide Ac-PhScN-NH₂ (8), with multiple-fold improved potency and enhanced systemic stability against endoproteases.⁶⁹

Figure 5.

Example of anticancer peptides with improved stability

Tachyplesin I (TPI, 9, Figure 6), a naturally occurring amphiphilic peptide, exhibits broad spectrum antimicrobial activity but was hemolytic against mammalian erythrocytes and susceptible to enzymatic degradation. To overcome these limitations, TPAD (10) was synthesized where all l-amino acids were replaced with corresponding D-amino acids and Ile¹¹ was substituted with D-Leu¹¹. The proteolytic stability examination after 2 hours of incubation with human serum indicated that more than 80% of 10 was present in the active state whereas around 80% of 9 was degraded. The 6 hours of incubation led to a complete degradation of 9 whereas 20% of 10 was available. With TPAD the percentage hemolysis became constant at concentrations above 100 μg/mL but with TPI a substantial increase was observed up to the highest tested concentration of 500 μg/mL.⁷⁰

Figure 6.

Structures of TPI and TPAD

hECP30 (11, Figure 7) exhibits potent antimicrobial activity but was susceptible to degradation by serine proteases due to the presence of six Arg residues, which act as potential targets of this enzyme (20% of the total sequence). In the quest to generate metabolically stable analog, L-ornithine (Orn) and l-proline (Pro) at the 1^st and 2^nd positions in the sequence were replaced with D-counterparts and the remaining five Arg residues were replaced with Orn (12). This modification improved the stability up to 30-fold by increasing the half-life to more than 480 min.⁷¹

Figure 7.

Structure of the antimicrobial peptide hECP30 and its modified analogs.

Overall, modulation of lipophilicity in DPDPE (1, Figure 2), by introducing two methyl groups on Tyr¹ to obtain Dmt, provides a fascinating outlook towards drug discovery by illustrating that minor changes in the canonical amino acids can lead to a significant change in the biological profile. Next, conversion of somatostatin (3, Figure 3) to octreotide represents a classic case of increasing the half-life of a natural hormone by developing of its analog with an unnatural amino acid (D-Trp⁸). The development of leucine-K59 from leucine-K3 depicts that minor change such as replacement of a single amino acid could lead to improved activity and metabolic stability. Lastly, PHSCN (7, Figure 5), TPI (9, Figure 6) and hECP30 (11, Figure 7) demonstrated that presence of unnatural amino acids leads to consequential changes in the pharmacodynamic characteristics of the peptides. Evidently, there are no specific patterns for modulating the lipophilic profile of peptides, but UAAs have proven as a highly effective tools to introduce desired lipophilic changes in the pharmacophore motif of clinically useful compounds.

3.2. Modulation of oral bioavailability

Peptides generally exhibit low oral absorption and bioavailability as compared to that expected of non-peptide drugs primarily due to pre-systemic enzymatic degradation and poor penetration across the intestinal membrane. We chose a few key examples to highlight the impact of UAAs on the oral bioavailability of peptides. Among the approaches, use of N-methylated amino acids for the enhancement of membrane permeability and oral bioavailability showed promising results.

Cyclo(Leu–Leu–Leu–Leu–Pro–Tyr) was initially selected for the backbone modification but showed low selectivity due to conformational incompatibilities. However, the replacement of L-Leu² and L-Pro⁵ with D-Leu² and D-Pro⁵ (13, Figure 8) improved the backbone N-methylation selectivity. The N-methylated peptide (14) showed improved permeability in the parallel artificial membrane permeability (PAMP) assay, almost equivalent to the orally bioavailable drug propranolol and 28% absolute oral bioavailability similar to cyclosporin A. The improvement in permeability is related to the peptide’s limited capacity to form H-bonds after N-methylation.⁷²

Figure 8.

N-methylated peptides with improved oral bioavailability.

Cyclic hexapeptide cyclo(-PFwKTF-) (15, Figure 8) acts on the somatostatin receptor with high selectivity towards subtypes sst2 and sst5. The hexapeptide 15 has poor oral bioavailability due to limited intestinal penetration and peptidase enzymatic breakdown. The N-methylated derivative 16 of cyclo(-PFwKTF-) exhibited enhanced intestinal permeability in the Caco-2 assay.⁷³

PMX53, Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg] (17, Figure 9) is a non-competitive complement C5a receptor 1 (C5aR1) inhibitor used to examine the role of C5aR1 in mouse models including central nervous disorders. PMX53 possesses an oral bioavailability of 9%. The replacement of the extracyclic Phe residue in PMX53 with a hydrocinnamate residue gave PMX205 hydrocinnamate-[Orn-Pro-D-Cha-Trp-Arg] (18), with improved oral bioavailability of 23% and better CNS penetration due to the enhanced lipophilicity. The subcutaneous injection of PMX205 resulted in high bioavailability (over 90%) with prolonged plasma and CNS exposure.^{74, 75}

Figure 9.

Structure of PMX53 and its analog

Leu-enkephalin, Tyr-Gly-Gly-Phe-Leu (19, Figure 10) is a selective agonist of δ-opioid receptor and plays a significant role in the treatment of chronic pain, inflammation, and cancer pain by avoiding respiratory depression and addiction. 19 exhibited poor pharmacokinetic profile due to rapid degradation by aminopeptidase at the Tyr¹-Gly² site. To improve the stability, the susceptible site was replaced with Tyr¹-ψ[(Z)CF=CH]-Gly² containing analog 20 that eliminated hydrogen bond donor and acceptor groups. In mouse plasma, both 19 and 20 displayed excellent stability and after 4 h. Conversely, in Sprague-Dawley rat plasma, 19 showed half-life of less than 5 min whereas 20 was 76% intact after 4 h. In human plasma, 19 showed a half-life of 12 min, while 68% of 20 remained available after 4 h. Peptide 20 exhibited improved stability in NADPH-charged microsomes and improved in vivo distribution and PK properties in BALB/c mouse model after iv administration.²⁵

Figure 10.

Leu-enkephalin and its fluorinated analog with improved profile

The bioavailability of an orally administered drug is governed by solubility, the capability to cross the intestinal mucosa, and metabolic stability. The uptake of a drug is regulated by either passive diffusion or through interactions with carrier proteins in the intestinal mucosa. Svenson and coworkers studied short cationic antimicrobial peptides derived from lactoferricin and investigated their transport via human intestinal dipeptide/tripeptide transporter 1 (hPEPT1). hPEPT1 mediated the efficient uptake of di and tripeptides in the mammalian intestine where they permeate passively across the cell membranes. The basic pharmacophore or structural requirement for the selected peptides included at least two hydrophobic residues along with two residues displaying positive charges (21, Figure 11). The introduction of hydrophobic unnatural amino acids and C-terminal amidation countered the transport capability for hPEPT1-mediated uptake as none of the peptides showed significant uptake. However, these active peptides showed high permeation via passive permeation identified using the PVPA study. Although, these peptides are not an active substrate for hPEPT1-mediated uptake, they still show high bioactivity and passive permeation making them potentially active for oral absorption.⁷⁶

Figure 11.

Structural features modulate the bioavailability of the lead peptide.

The examples discussed above provide insight into approaches stability approaches. Peptides 13 and 15 (Figure 8) show that minor changes such as masking H-bonding by N-methylation can lead to improved oral bioavailability. However, in PMX53 (17, Figure 9) replacement of the canonical Phe residue with a hydrocinnamate residue delivered improved oral bioavailability and better CNS penetration. Metabolically susceptible site of Leu-enkephalin (19, Figure 10) is chemically changed to fluoroalkene, that stabilizes the cleavage site and provides an improved bioavailability profile. In a nutshell, peptide oral bioavailability is a multidimensional property, as there are diverse underlying reasons for low bioavailability. Importantly, high oral bioavailability can be achieved through multiple ways, and UAAs have played an important role in optimizing oral bioavailability. However, peptides need to be carefully analyzed before carrying out chemical modifications.

3.3. Modulation of permeability

Permeability poses a significant challenge in peptide-based drugs due to their high molecular weight and large size. One of the biggest obstacles in the design and synthesis of linear and cyclic peptide drugs is to attain high membrane permeability. UAA incorporation can improve the permeability of peptides through modulation of physicochemical and ADME properties. A demonstration of this approach was exhibited in a library of cyclic peptides incorporating γ-, D-, and N-methylated residues into cyclic peptide-polyketide constructs. The cyclic peptide cyclo(D-Pro-S,S-Sta-D-MeLeu-L-MeLeu-S,S-Sta-L-MeLeu) (22, Figure 12) displayed good live-cell permeation and better metabolic stability in human liver microsomes. During the In vivo study, analog 22 demonstrated an enhanced ADME profile with good bioavailability showing AUC values of 1697 and 1760 ng.h.mL^-1 upon iv and oral administration, respectively.⁷⁷

Figure 12.

Structure of short cyclic peptide containing statine

In a quest to find orally bioavailable peptides for a variety of therapeutically relevant targets, a in silico tool has been developed for designing macrocyclic peptides. Three-dimensional descriptors like the free energy of transfer from water to membrane for a macrocycle in its lowest energy conformation (ΔG*_transfer), lipophilicity, and polar surface area were found to be reliable predictors of permeability and oral exposure for macrocycles closed by thioether linkages. Specifically, N-methylated peptides proved effective at limiting solvent-exposed hydrogen bond donors and presumably lowering the desolvation penalties associated with entering a membrane. For example, the unmethylated parent peptide 23, (Figure 13) had three solvent-exposed backbone NHs and a calculated ΔG*_transfer value of −4.3 kcal.mol⁻¹ resulting in undetectable permeability in a PAMPA assay with log P_app< −5.9. Alternatively, N-methylated analog 24, was predicted to have no solvent-exposed NHs in its lowest energy conformation and showed a 1.95 kcal.mol⁻¹ lower ΔG*_transfer (−6.25 kcal.mol^-1) relative to 23. The subsequent PAMPA assay revealed a favorable permeability of 24 with a log P_app value of −4.3. In silico passive permeability and oral exposure prediction data were comparable with in vitro and in vivo findings.⁷⁸

Figure 13.

N-methylated thioether containing cyclized peptide.

These examples reveal that the permeability of peptides can be successfully improved by strategically placing unnatural amino acids in the peptidic sequence of newly discovered compounds. Occasionally, multiple chemical changes are also required along with the installation of these amino acids to obtain a favorable pharmacological profile.

Researchers have studied various linear and cyclic peptides and delivered clinically useful agents. To bring medicinally useful changes in peptide molecules, peptide compounds can be chemically edited to have a considerable impact on pharmacological profile and physicochemical properties such as solubility and membrane permeability, potency, and selectivity of drugs, thereby improving their biodistribution, pharmacodynamics, and toxicological profile. Importantly, modulating the physicochemical properties profile of peptides has no specific patterns, but scientists need to carefully assess the biological targets, metabolic sites, peptidases, and nature of pharmacophore motifs to carry out selected chemical changes through unnatural amino acids in peptides, which can lead to clinically useful compounds.

4. Recent designs and developments of unnatural amino acid containing analogs in medicinal chemistry and drug discovery

Unnatural or non-canonical amino acids also appear in peptide natural products, specifically non-ribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs). Accordingly, UAAs have been an area of great interest in peptide or non-peptides based drug discovery for wide range of therapeutic targets and disease areas. We conducted an extensive survey of published articles highlighting UAAs in drug discovery and medicinal chemistry research. Several filters based on the biological properties, profiles of peptides and types of UAAs were applied before segregating the most relevant literature to discuss in this perspective. We delineated suitable articles and categorized them on the basis of therapeutic targets and related disease areas.

4.1. Unnatural amino acids in anticancer agents

As discussed in the previous section, the properties of native peptides and small drug molecules can be altered by unnatural amino acids. This section is focused on the role of unnatural amino acids in the development of new classes of anticancer agents, such as SETD8 inhibitors, STAT inhibitors, arginase inhibitors, carborane-based agents, and modifications to an existing therapeutic agent. UUAs can provide more potent and less toxic anticancer drugs such as antibody-drug conjugates, coibamide A, and gossypol derivatives.

Folate receptor antagonists:

Folates and antifolates are transported across the membranes via various transporters including folate receptors (FRs), reduced folate carrier (RFC), and proton-coupled folate transporter (PCFT), which are over-expressed in tumor cells and serve as important targets in anticancer research. 2-Amino-4-oxo-pyrrolo[2,3-d]pyrimidine (26),⁷⁹ an analog of antifolate compound pemetrexed (25, Figure 14),⁸⁰ was obtained by substituting the 6-position with 3-C bridge attached to glutamate residue. Selective FRs analogs of 26 were synthesized by substituting the terminal amino acid, L-glutamate, with natural and unnatural amino acids and evaluated for anticancer activity. Compound 26 expressed significantly improved activities with IC₅₀s of 0.31, 0.17, and 3.34 nM against FRα, FRβ, and PCFT, respectively, in CHO cells and IC₅₀ of 0.26 nM against KB human cancer. Compound 27 showed IC₅₀s of 9.5 and 3.6 nM against FRα and FRβ, respectively, in CHO cells and IC₅₀ of 9 nM in KB cells. Modified analog 27 exhibits higher selectivity for FRs over other uptakes and could be used in targeted chemotherapy with reduced dose-related toxicity.⁷⁹

Figure 14.

Analogs of pemetrexed as folate receptor antagonists

Anti-FRs:

The small cytotoxic drugs used in cancer chemotherapy to halt the proliferation of rapidly growing cells are associated with profound systemic toxicity due to non-specifically accumulated drugs in the healthy organs. Conjugating small drugs with monoclonal antibodies to generate antibody-drug conjugates (ADCs) can address this issue because the antibody component can act as a delivery system with target specificity.⁸¹ An anti-FRs ADC DGN549 (28, Figure 15) of potent DNA alkylator indolinobenzodiazepine is catabolized to a very potent anilino metabolite 29 via cleavage of the amide bond between the L-alanyl residue of the dipeptide (L-Ala-L-Ala) and aniline ring. The metabolite 29 is responsible for the bystander killing of antigen-negative cells, which increases the antitumor activity.⁸² In a recent report, the effect of a change in the stereochemistry of amino acids was demonstrated in DGN549 ADC on antitumor efficacy using four diastereomers of dipeptide i.e., L,L (30), D,L (31), L,D (32), and D,D (33) conjugated with antibodies. All four ADC derivatives were evaluated in vitro and in vivo for tolerability and efficacy and exhibited potency in high-antigen-expressing KB and T47D cell lines with IC₅₀ ranging between 5 and 40 pM. In the low-antigen-expressing cell line NCI-H2110, analog 30 (IC₅₀ = 80 pM) exhibited 3-fold increased activity compared to 31 and 32 and 90-fold higher potency than 33. This study demonstrates that analogs with the natural linker L,L were easily cleaved by peptidase, resulting in the active metabolite 29 that produced high potency in H2110 cells, whereas in the case of unnatural analogs 31 and 33, the formation of 29 was very slow. Bystander activity of all diastereomers was determined by using a mixture of FRα-positive and FRα-negative 300.19 cells, which showed the selective killing of antigen-positive cells. In vivo studies using NCI-H2110 xenograft revealed that analog 30 exhibited anti-tumor activity at a low dose of 0.3 mg/kg whereas 31 and 33 were unable to produce bystander activity, and 31 showed anti-tumor activity at a high dose of 1.5 mg/kg.⁸³

Figure 15.

Indolino benzodiazepine antibody-drug conjugates (ADCs)

SETD8 inhibitors:

The methylated histone H4 at Lys-20 showed involvement in cell division processes such as mitotic regulation, DNA replication, and damage repair. SETD8, a 352 amino acid containing methyltransferase, catalyzes site-specific monomethylation of the ɛ-amino group at Lys-20 in histone H4, KRHRK²⁰VLR (34, Figure 16).⁸⁴ SETD8 is overexpressed in breast cancer, lung carcinoma, hepatopancreatic carcinoma, bladder cancer, and chronic myelogenous leukemia. Therefore, targeting SETD8 is a promising target for developing anticancer agents, and 34 was utilized as a template to design potent SETD8 inhibitors. The crystal structure of H4 bound to SETD8 revealed that the Y245 residue side chain is in proximity to the ɛ-amino group of Lys (K-20) surrounded by hydrophobic pockets that energetically disfavored the insertion of K20. The replacement of Lys (K-20) with natural and unnatural amino acids was performed using molecular modeling to develop SETD8 inhibitors that exhibit favorable interactions with hydrophobic side chains. The peptide substituted with norleucine (KRHRNleVLR, 35) exhibited strong binding (K_d = 0.14 μM, IC₅₀ = 0.33 μM, K_i = 50 nM) against histone H4 peptide and SAM. Peptide 35 also selectively inhibited STED8 when tested against 32 different methyltransferases.⁸⁵

Figure 16.

Mutation of the lysine residue of the H4 peptide using unusual amino acid norleucine for the improvement of the binding to the anti-cancer target STED8

Sec61α translocon inhibitors:

Coibamide A (36, Figure 17, IC₅₀ = 1.4 nM) is a naturally occurring macrocyclic N-methylated depsipeptide that exhibits potent antiproliferative activity.⁸⁶ Coibamide A inhibits Sec61α translocon, a channel transporter that controls the translocation of regulatory proteins and is a potential anticancer target. In coibamide A, the labile ester linkage between L-MeThr⁵ and D-MeAla¹¹ was replaced with MeLys(Me) to obtain analogs with improved molecular stability. Furthermore, amino acids of MeSer(Me)³ and MeSer(Me)⁶ were replaced with MeAla, leading to peptide 37, which exhibited a 300-fold decrease in potency. Derivatives of 37 were synthesized via modifications such as the replacement of MeLys(Me) with amino acids of varied length and stereochemistry. Replacement of L-Tyr(Me)¹⁰ with L-β-(4-biphenyl)alanine (Bph) in 37 gave a peptide with seven-fold greater bioactivity (IC₅₀ = 0.060 μM). To analyze the impact of Bph, various analogs were synthesized by replacing L-Tyr(Me) in 36 with Bph. Peptide 38 demonstrated 12-fold greater efficacy (IC₅₀ = 0.11 nM), and peptide 39 with MeAla at the 3^rd and 6^th positions also exhibited significant bioactivity (IC₅₀ = 0.25 nM).⁸⁷

Figure 17.

Coibamide A mimetics

Pan-Ras inhibitors:

The oncoprotein Ras, a subfamily of small GTPases, is one of the most challenging targets in cancer chemotherapy due to its flat structure and lack of any major binding sites for small molecules. Generally, K-Ras (Ras isoform) and GTPase work in coordination during the cell signaling process, and activation of K-Ras leads to an increase in the GTP level and the dissociation of GDP. GTPase initiates the hydrolysis of GTP, which leads to further signaling cascades terminated due to reduced Ras activity. Mutation in the Ras protein enables excessive cell proliferation and durability due to inhibition of GTPase activity. Cyclorasin B3 (40, Figure 18), a bicyclic peptide, was identified as a Ras inhibitor (K_D = 1.6 μM) that induces death in cancer cells via apoptosis.⁸⁸ The optimizations carried out on 40 led to molecules with improved selectivity, potency, and cell permeability. To begin, D-Val was replaced with D-Arg at the 9^th position, along with the inclusion of linker β-Ala–β-Ala–Lys at the 11^th position. An alanine scan showed that Arg at the 8^th position is important for binding to Ras. In search of better analogs, the exocyclic linker at the C-terminal was replaced with unnatural amino acids. Peptide B4 (41) with the linker (Orn–D-Arg–Fpa–D-Ala-Lys) showed inhibitory activity in the HTRF assay (IC₅₀ = 1.7 μM) and an EC₅₀ of 17 μM in the MTT assay against H358 cells. The truncation of C-terminal residues (Orn–D-Arg–Fpa–D-Ala-Lys) was performed to reveal that removal of L-Lys improves Ras binding and cellular activity, whereas truncation of D-Ala results in loss of cellular activity. The truncation of both D-Arg and Fpa reduced binding and cellular activity, indicating their significance. After several attempts, peptide B4-27 (42) was identified (IC₅₀ = 0.69 μM) in the HTRF assay and (EC₅₀ = 2.1 μM) in the CellTiter-Glo assay, and 76-fold more potency than 40. B4-27 (42) showed improved cell permeability, as confirmed by the confocal images, and high metabolic stability (t_1/2> 24 h). Immunohistochemistry (IHC) showed a remarkable reduction in ERK phosphorylation, indicating reduced tumor growth in mouse xenografts at a dose of ≤5 mg/kg.⁸⁹

Figure 18.

Cyclorasin-based Ras inhibitors

Other anticancer strategies:

Carborane is a boron-rich polyhedron with anticancer potential against human brain tumors through boron neutron capture therapy (BNCT). In BNCT, thermal neutron irradiation results in the nuclear fission of isotope ¹⁰B and the release of α-particles, resulting in cell death.⁹⁰ The impact of S-phenyl-L-cysteine-based derivatives linked to carborane was studied in the human glioblastoma cell line U87 (43, Figure 19). Upon exposure to cysteine-conjugated carborane, i.e., 2-amino-3-(1,7-dicarba-closo-dodecaboranyl-1-thio)propanoic acid (44), cells exhibited morphological alterations within 5 minutes of exposure that persisted even after 48 h of treatment. In the absence of radiation, exposure of 44 at 1 μM to 1000 μM in the cells exerts a strong cytostatic effect with 50% decrease in cell population at the 48 h time point; however, no change was observed in U87 cells treated at concentrations lower than 1μM. qPCR analysis revealed a 5-fold downregulation of gene expression, which is associated with cell cycle and proliferation, and an upregulation of genes associated with the apoptosis pathway. The flow cytometric analysis confirmed the cell cycle arrest in M phase due to depression of cyclin-dependent kinases (CDKs), polo-like kinase 1 (PLK 1), and retinoblastoma protein (RBL1) 48 h post-treatment of cells with 44 at 1 mM concentration. The rapid uptake and prolonged effect of 44 on cancer cells without radiation suggested further investigations to study its effect on cancer cells with irradiation.⁹¹

Figure 19.

Substituted S-cysteine derivatives

Minigastrin (MG)-inspired peptides are a potential ligand of the cholecystokinin-2 receptor, a target expressed in tumors including medullary thyroid carcinoma (MTC). Despite having a higher tumor uptake value, MG analogs show low metabolic stability and high renal accumulation. MG analogs were developed by site-specific modification at the C-terminus of the receptor binding sequence of minigastrin. Modified C-termini with unnatural aromatic amino acids and N-methylated amino acids, and an additional proline residue at the N-termini led to improved metabolic stability of the peptides and a favored tumor-kidney ratio. Replacement of Met with N-Me-norleucine and Phe at position 13^th with 1-naphthylalanine (1-Nal) resulted in MGS5, a potent CCK2R binding ligand with in vivo enzymatic stability. Preclinically, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA-conjugated MGS5 45 (Figure 20) labeled with radionuclide was investigated for molecular imaging and targeted radiotherapy. Additional inclusion of Pro residue at the N-terminus of MGS5 resulted in improved tumor cell uptake and increased metabolic stability. Peptide [¹⁷⁷Lu]Lu-DOTA-1(46) showed an uptake value of 34% (IA/g) for A431-CCK2R tumor xenograft after 4 h p.i. as compared to 20% (IA/g) for parent 45 in BALB/c mice. In vivo enzymatic stability studies showed that 57-79% of the cell remained unchanged after 1 h of incubation in the blood of BALB/c nude mice, which is comparable to that of 45 (70%). Notably, 46 showed a high tumor kidney ratio of 10 as compared to 45 (4-6), highlighting its clinical relevance in molecular imaging of tumor-expressing cells.^{92, 93}

Figure 20.

Radiolabeled minigastrin analogs

In cancer research, diverse UAAs have been explored which have aided in improving the therapeutic profiles of peptides. Optimization of peptide 35 (Figure 16) gave the desired activity against 32 different methyltransferases in a linear peptide. The development of Cyclorasin-based Ras inhibitor (42 Figure 18) involves the strategic use of multiple UAAs. Interestingly, compound 44 (Figure 19), a cysteine-conjugated carborane, was obtained by a single chemical modification. In the emerging area of cancer targeting of radionuclides and targeted radiotherapy, the development of peptide 46 from MGS5 45 (Figure 20) with improved tumor cell uptake and metabolic stability was achieved by multiple UAAa in its motif. These examples of UAAs showed that therapeutic development of anti-cancer agents can be successful, and peptides can be optimized for the desired therapeutic profile. The optimization of complex drug targets can be challenging and require extensive chemical modifications, but a favorable profile is achievable.

4.2. Unnatural amino acids in neurological and neurodegenerative (CNS) disorders

4.2.1. Analgesics

Common treatment for pain is based on non-steroidal anti-inflammatory drugs (NSAIDs) and opioids. There are limited options for chronic pain treatment, and opioids are efficacious analgesics but are associated with addiction, tolerance, and respiratory depression. Therefore, medicinal chemists are continuously looking for new classes of agents. FDA-approved drugs for pain treatment that feature unnatural amino acids include pregabalin and ropivacaine. Unnatural amino acids are combined with peptides and small molecule analgesic agents to improve pharmacological parameters and are being actively investigated as opioid receptor agonists, neurotensin agonists, and neuropeptide FF receptor antagonists.

4.2.1.1. Opioid receptor agents

μ-opioid receptor agonists:

Unnatural amino acid sarcosine (Sar, N-methylglycine) containing peptide Tyr-D-Arg-Phe-Sar (47, Figure 21), also termed TAPS, exhibited analgesic action via activation of the μ₁-opioid receptor (MOR) but showed poor metabolic stability. Later, TAPS was cyclized to improve stability and a TAPS c(n-m) library was prepared, where n and m represent the lengths of the alkyl chains on the amide nitrogen of glycine and arginine, respectively. The studies revealed that TAPS c(2-6) (48) produced significant MOR agonistic activity with 48% activation in CHO cells expressing MORs without any antagonism. Rat origin pooled brush border membrane vesicle (BBMV) enzymatic degradation study showed an overall increase in metabolic stability of 48 with no change in concentration up to 120 minutes. The intraperitoneal (ip) administration of 48 at a 15 mg/kg dose resulted in a prolonged analgesic effect similar to that with reference morphine (5 mg/kg). The Caco-2 assay of 48 showed a low permeability coefficient (P_app) value of 3.8 x 10^-8 cm/s. These results highlight 48 as a peripheral painkiller free from the centrally acting opioid-related side effects. In a mouse open field test (OF test), the animals administered with 48 (15 mg/kg, ip) showed the absence of any locomotor activity, confirming its peripheral activity.⁹⁴

Figure 21.

Peptides as selective MOR agonists

Substituted tyrosine analogs containing a tetrahydroquinoline (THQ) moiety at the N-terminus showed opioid agonistic activities, and slight modifications generated MOR-selective analogs (Figure 21). [³⁵S]-GTPγS binding assay revealed that peptide with Dmt residue (49, Figure 21) exhibited promising affinities at opioid receptor subtypes: MOR at 0.22 nM, δ-opioid DOR at 9.4 nM, and KOR at 68 nM. The peptide 49 showed high agonistic potential at MOR with an EC₅₀ of 1.6 nM and 81% maximal stimulation compared to standard agonist DAMGO but much weaker activity on DOR and KOR. Replacement of the methyl group with chlorine at the 2- and 6-positions of tyrosine gave 50, which exhibited a 20-fold lowered potency in the [³⁵S]GTPγS assay compared to 49 (EC₅₀ = 33 nM) along with 83% stimulation of MOR. Altogether, Dmt is the key building block for developing new opioid peptidomimetic with improved biological activity.⁹⁵

Constrained dermorphin-based analogs were obtained by modifying the mixed opioid agonist H-Dmt-D-Arg-Aba-Gly-NH₂ (51, Figure 22) as an opioid receptor ligand. Incorporation of conformationally constrained unnatural amino acids such as 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one (Dhq), and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana) in peptide 51 resulted in a significant change in selectivity and functional activity at both MOR and DOR. Insertion of the 2-Ana-Gly unit in 51 gave peptide 52, which showed higher potency (IC₅₀ = 21.4 nM) and better binding affinity (IC₅₀ = 2.19 nM) for MOR in the GPI assay. The isolated MVD test showed a significant decrease in DOR binding affinity (IC₅₀ = 153 nM), which resulted in moderate DOR agonism (IC₅₀ = 114 nM). The replacement of D-Arg² in 52 with D-Ala² gave opioid antagonist 53 with potent MOR with weak DOR inhibitory activity (IC₅₀ = 2.88 nM and 306 nM, respectively). These constrained building blocks helped modulate opioid receptor selectivity and potency by optimizing the spatial orientation of the peptides.⁹⁶

Figure 22.

Conformationally constrained dermorphin-based tetrapeptide analogs

MOR/DOR agonists and KOR antagonists:

Linear enkephalin-like tetrapeptide (H-Tyr-Gly-Gly-Phe-OH) analogs act as a multifunctional ligand of the opioid receptors with improved bioavailability. N-Phenyl-N-piperidine-4-yl-propionamide (Ppp), a lipophilic pharmacophore of fentanyl was incorporated at the C-terminus of the parent peptide. The modified peptide Dmt-D-Ala-Gly-Phe-Ppp (54, Figure 23) produced mixed μ/δ agonistic activity with K_i values of 0.36 nM (hDOR), 0.38 nM (rMOR), and 21 nM (hKOR). To further optimize 54, homologation at Ala² and halogenation of Phe⁴ was executed.⁹⁷ LYS744 (55), containing Nle and 4-chloro-Phe residues at positions 2 and 4, respectively, exhibited binding affinity to MOR and DOR with K_i values of 0.10 and 0.08 nM. In vitro, 55 showed potent opioid agonistic activity with IC₅₀ values of 1.3 nM and 1.9 nM in the GPI and MVD assays. In the [³⁵S] GTPγS assay, 55 showed good efficacies (EC₅₀ = 0.14 nM and 0.07 nM in MOR and DOR). A spine nerve ligation (SNL) study in rats revealed antinociceptive properties of 55 upon intrathecal (it) administration (3-30 μg/5μL). The analog 55 also exhibited binding affinity at KOR with K_i of 1.3 nM and full antagonist activity at KOR (IC₅₀ = 52.4 nM) with an I_max of 122%. Although compound 55 retained its stability in plasma even after 42 days of incubation, the high efflux ratio of around 2.17 and low apparent permeability (Papp) of 0.38-0.83 x 10^-6 cm/s made it a poor candidate for BBB crossing.⁹⁸

Figure 23.

N-phenyl-N-piperidine-4-yl-N-propionamide (Ppp) containing enkephalin derivatives

4.2.1.2. Other receptors

Neurotensin Agonists:

Neurotensin (NT), a peptide-based endogenous neuromodulator, is a widely investigated ligand due to its antinociceptive effect generated by the activation of non-opioid receptors, NTS1 and NTS2.⁹⁹ In silico investigations demonstrated that the C-terminal part of neurotensin, NT(8-13), H-Arg-Arg-Pro-Tyr-Ile-Leu-OH (56, Figure 24), effectively binds to NTS1. This motif has been modified to develop neurotensin analogs with an improved half-life and high selectivity towards NTS2.¹⁰⁰ Neurotensin analogs with selective binding to NTS2 were synthesized. Three predominant sites of cleavage by proteolytic enzymes, viz. Arg⁸-Arg⁹, Pro¹⁰-Tyr¹¹, and Tyr¹¹-Ile¹², were determined in NT, and modifications were performed. The Ile¹² was replaced by tert-leucine (Tle), and it improved stability and enhanced selective binding to NTS2. Another modification concerned replacing the Tyr¹¹ with D-Trp, Dmt, meta-tyrosine (mTyr), or 6/7-hydroxy-L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids (6-OH-Tic or 7-OH-Tic) [and then what was the result?]. To improve the pharmacological properties, the Arg^8,9 residues were replaced by Lys. The Lys⁸ residue was further replaced by β³-homolysine, which gave the best binding affinity at NTS2 (K_i = 0.11 nM) and was 89.3-fold selective towards NTS2. The NT analog β³-hLys⁸-Lys-Pro-(6-OH)Tic¹¹-Tle¹²-Leu (57) exhibited 1324-fold selectivity towards NTS2 with binding affinity (K_i = 2.9 nM) and high metabolic stability (half-life >24 h). The in vivo experiments on 57 revealed a strong analgesic effect by inhibiting the formalin-induced pain with an ED₅₀ of 1.4 nmol and no significant hypotension (or) hypothermia.¹⁰¹

Figure 24.

Structure of the Neurotensin and its analogs.

Chimeric opioid-neurotensin analogs:

A series of potent bifunctional chimeric decapeptides were obtained by merging the non-opioid pharmacophore of neurotensin with the opioid pharmacophore based on the lead PK20 (58, Figure 25).¹⁰² Peptide 58 induced analgesia through both central and peripheral action by activating MOR and NTS.¹⁰³ The opioid-neurotensin (OP-NT) decapeptides were obtained by fusing the C-terminal β-Ala unit of the dermorphin-derived opioid with N-terminus of the NT_8-13 analogs. Among synthesized peptides, 59 was found to be the most active chimer with effective binding affinity towards MOR (K_i = 1.75 nM), NTS2 (K_i = 0.003 nM), and NTS1 (K_i = 1.75 nM) receptors. The presence of Tle improved enzymatic stability, and incorporation of β³hArg and Dmt residues improved the NTS2 selectivity of the analogs. Peptide 59 showed NTS1 activation (EC₅₀ = 0.86 nM) and was an agonist of MOR G-protein activation (EC₅₀ = 28.7 nM) in the [³⁵S]GTPγS binding assay. Furthermore, 59 was found to be less effective against MOR-mediated β-arrestin-2 signaling (EC₅₀ = 1066 nM) in the PathHunter assay, depicting its safer analgesic action. In the tail-flick test in mice, 59 (2.64 μmol/kg, sc) displayed long lasting antinociceptive efficacy for 8 h with more than 80% of the maximum possible effect for 2 to 7 h, and excellent stability in human plasma with a half-life of 467 minutes.¹⁰²

Figure 25.

Neurotensin analogs.

4.2.2. Alzheimer’s disease

Alzheimer’s disease is a neurodegenerative disorder in older individuals marked by a decline in memory and cognition and associated with the accumulation of Aβ amyloid plaques in the brain. The most promising therapies involve agents that interfere with the accumulation of Aβ aggregates. Aβ plaques are made up of misfolded proteins; therefore, peptides are being investigated as amyloid-β (Aβ) aggregation inhibitors.

Amyloid-β (Aβ) aggregation inhibitors:

Inhibition of Aβ aggregates is a promising approach for developing therapeutics to treat Alzheimer’s disease. A series of linear tetrapeptides containing unnatural amino acids,¹⁰⁴ inspired by the lead sequence of the parent Aβ_39-42 fragment (Val-Val-Ile-Ala-OH, 60, Figure 26),¹⁰⁵ were developed as potential amyloid-β (Aβ) aggregation inhibitors. The C-terminus of 60 was amidated, and the N-terminal Val was replaced with D-Phe to give D-Phe-Val-Ile-Ala-NH₂ (61). Peptide 61 exhibited 100% cell viability at tested 10 μM concentration against Aβ aggregation-induced neurotoxicity in the MTT cell viability assay and 100% inhibition of Aβ aggregation in the thioflavin-T fluorescence (ThT) assay at 2 μM. Peptide 61 showed high BBB permeation (P_e = 5 x 10^-6 cm/s) and metabolic stability up to 12 h and 24 h of serum and trypsin treatment, respectively. SAR studies suggested that amidation of 60 results in alteration of the activity and improved proteolytic stability, and the altered conformation and hydrophobic amino acid replacement of residues at the N-terminal affect the Aβ aggregation.¹⁰⁴ Further, linear tetrapeptides based upon Aβ_39-42 containing isosterically analogous unnatural or natural amino acids were developed. Replacement of Phe with 4-F-Phe in 61 gave 62, which displayed potent inhibition of Aβ aggregation with 83.1% cell viability even at a low concentration of 0.5 μM. Similarly, the replacement of Val⁴⁰ with octahydroindole-2-carboxylic acid (Oic) gave 63 (Val-Oic-Ile-Ala-OH), which displayed 84.9% cell viability at 0.1 μM in the MTT assay and 61.2% Aβ aggregation inhibition in the ThT assay. Both Aβ_39-42 analogs, 62 and 63, showed comparative BBB permeability i.e., more than 4 x 10^-6 cm/s, and serum stability of more than 90% upon 12 h of treatment.¹⁰⁶

Figure 26.

Peptide-based β-amyloid inhibitors.

4.2.3. Other CNS active agents

GABA-AT inhibitors:

γ-Aminobutyric acid aminotransferase (GABA-AT) is a promising therapeutic target for epilepsy and cocaine addiction treatment.¹⁰⁷ Cyclopentyl γ-amino-acid analogs showed potent GABA-AT inhibitory activity (Figure 27). Specifically, FCP, (1R,3S,4S)-3-amino-4-fluorocyclopentane carboxylic acid 64 displayed significant GABA-AT binding affinity with a K_i value of 0.053 mM but a lower maximum inactivation rate (K_inact) of 0.011 min^-1 along with a limited inactivation efficiency (K_inact/K_i) of 0.20 min^-1mM^-1 compared to the standard anticonvulsant drug vigabatrin. Supported by computational analysis, the incorporation of an endocyclic double bond in 65 was hypothesized to bring the fluorinated group much closer to the Lys329 residue in the binding site of GABA-AT, which improved the inactivation rate and enhanced the K_inact. Accordingly, 65 produced a 2-fold enhancement of binding affinity (K_{i =} 0.026 mM), a 12-fold increase in rate constant (K_inact= 0.132 min^-1) and 25-times more efficient inhibition of GABA-AT compared to 64. Furthermore, relative selectivity of GABA-AT over other aminotransferases such as aspartate- and alanine-transferases by 65 suggested its importance as a potent and efficient inactivator of GABA-AT.¹⁰⁸

Figure 27.

GABA-AT inhibitors based on unnatural amino acids.

4.3. Unnatural and modified amino acids in anti-infective agents

The development of multidrug-resistant strains and superbugs is an area of increasing concern in global health. Anti-infective peptides and small molecules with unnatural amino acids provide unique mechanisms of action and can target sites associated with membrane disruption and lysis, making them less prone to mutational forms of resistance associated with protein targets. The FDA has approved many drugs including micafungin, daptomycin, telavancin, and plazomicin with unnatural amino acids that impart drug-like properties as potent, metabolically stable, and less cytotoxic therapeutics.

4.3.1. Antibacterial agents

The discovery and development of antibacterial agents were initiated by the conjugation of several classes of agents with unnatural amino acids. The most prominent among these classes are lipopeptides such as stalobacin I and polymyxin B analogs. Alterations in already existing antibiotics such as daptomycin, feleucin, and nisin have generated more potent analogs with improved physicochemical properties.

Lipopeptide antibiotics:

Stalobacin I (66, Figure 28) is a potent lipopeptide-based antibiotic with two different peptidic sequences bridged by a hemiaminal methylene group. Stalobacin I was discovered while investigating the metabolites of an unknown Gram-(-) bacteria, PBJ-5360 and exhibited potent antibacterial activity with an identical MIC value of 0.004 μg/mL against Staphylococcus aureus, Enterococcus faecalis, E. faecium, and S. pneumoniae, along with MICs of 1 and 4 μg/mL against Escherichia coli and Klebsiella pneumoniae. Stalobacin I (66) showed 125-250 fold stronger activity against S. aureus than the existing antibiotics, including vancomycin, linezolid, and telavancin. The peptide acts by instigating characteristic morphological changes in the cellular structure of bacteria. The structure elucidation of Stalobacin I confirmed the presence of unnatural amino acids such as carnosadine, 3,4-dihydroxyarginine (3,4-diHyArg), 3-hydroxyisoleucine (HyIle), and 3-hydroxyaspartic acid (HyAsp), along with a cyclopropyl fatty acid chain in its sequence. The sequence of amino acids in the lower half was deduced to be Asp-3,4-diHyArg-HyIle-Gly-Ser. The upper half possessed HyAsp-N-MeAsp amino acids with an amidated N-terminal to the 2-hydroxy-3-butenoic acid moiety, whereas the C-terminal was linked with the carnosadine lactam moiety via an amide linkage.¹⁰⁹

Figure 28.

Structure of Stalobacin I

Polymyxin B (67, Figure 29) is a cyclic lipopeptide effective against Gram-(−) bacteria, which acts by interacting with the lipopolysaccharides of the outermost membrane. The next generation polymyxin analogs were explored due to the declining susceptibility of 67 to resistant strains, and the side effects associated with overuse. The unnatural amino acid residue diaminobutyric acid (Dab) plays a significant role in interacting with the lipopolysaccharide membrane of the bacteria to elicit bactericidal activity.¹¹⁰ The replacement of the fatty acyl side chain and the 1^st amino acid residue in 67 with the β-arylated aminobutyric acid (a substituted Dab residue) gave peptide (68) that exhibit MICs of 0.06-0.25 μg/mL against E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii with reduced cytotoxicity and nephrotoxicity.¹¹¹ Then D-Phe⁶ was replaced with 4-bromophenylalanine (69), biphenylalanine (70), and 4-pyridylalanine (71) that showed MICs of 0.5-8 μg/mL against E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. Another modification was done by replacing D-Phe with cyclohexylalanine (Cha) (72), which showed the MICs in the same range. Though these modifications improved the antibacterial activity of polymyxin analogs, cytotoxicity was not reduced owing to the increased hydrophobicity.¹¹² Later, a next-generation polymyxin candidate with the ability to act as a potentiator of rifampicin was developed.¹¹³

Figure 29.

Structure of Polymyxin and the modified analogs.

The antibacterial activity of constraint cyclic peptides can be modified by incorporating flexible amino acids such as ω-amino and α,ω-diamino acids. A library of cyclic heptapeptides comprised of all α-amino acids (homodetic) was reported with glycine or unnatural amino acids at the C-terminus. One peptide with Gly⁷ at the C-terminal, LR(2-Nal)LR(2-Nal)G (73, Figure 30), demonstrated better efficacy with identical MICs of 8 μg/mL against MRSA and VRE and 16 μg/mL against Candida albicans, along with non-hemolytic nature (EC₅₀ = 215 μg/mL). However, the efficacy of the cyclic peptide was enhanced when Gly⁷ was substituted with ω-amino and α,ω-diamino acids. Peptide 74 bearing 8-aminooctanoic acid (8-Aoc⁷) exhibited MIC value of 2 μg/mL against both MRSA and VRE and 16, 8, 16, and 4 μg/mL against E. coli, A. baumannii, K. pneumoniae, and C. albicans. Molecular mechanics calculations in conjunction with CD experiments established that the installation of flexible ω-amino and α,ω-diamino acids led to an improve antimicrobial profile.¹¹⁴ Later, a macrocyclic peptide 75 was identified from a combinatorial library and synthesized by replacing the Gly with an array of flexible ω- and α,ω-diamino acids. The alkyl chain length was varied up to 5 carbons, introducing lipophilic linkers into the macrocyclic scaffold, and analogs were developed using polar and cationic linkers. Antibacterial activity was retained for most of the peptides specifically against MRSA and VRE, where analogs with 4-Abu (76) and Dab (77) linkers showed MICs in the range of 4-16 μg/mL and 2-8 μg/mL, respectively. The modified peptides (76-77) exhibited antibacterial activities similar to those of 75, but were faster acting.

Figure 30.

Structure of the flexible ω-amino acid and α,ω-diamino acid containing cyclic peptides.

The lantibiotics are peptides synthesized by bacterial ribosomes, composed of unnatural amino acids lanthionine (Lan) and Me-lanthionine (MeLan). Nisin, a broad spectrum lantibiotic, has been widely explored owing to its highly selective lipid-II binding affinity. However, it lacks drug-likeness due to poor aqueous solubility and enzymatic degradation. Nisin bioconjugate lantibiotic (78, Figure 31) was semi-synthetically developed via a flexible linker. Nisin fragment 1-12 (A+B) was utilized, as the lipid II binding domain, and the pore-forming peptide was the pexiganan and both domains were linked through the PEG4 linker. The lantibiotic 78 when tested for antimicrobial activity, had comparable (MIC = 0.75 μM) but lower activity than nisin (MIC = 0.0234 μM) against Micrococcus luteus. It was observed that the bioconjugate had a better pharmacological profile compared to the nisin with good aqueous solubility and greater stability.¹¹⁵

Figure 31.

Structure of the Nisin bioconjugate.

A series of peptides were synthesized by modifying the length of the alkyl chain of unnatural lipoamino acids (LAAs) and the number of lysine residues in linear and cyclic forms. The peptides were tested, and lipopeptide (79, Figure 32) was identified as the most active peptide indicating two lysine residues and LAA C₁₂ were optimum for significant activity. Lipopeptide (79) showed selective inhibition with MICs in the range of 0.06–0.2 μM against VRSA, MRSA, and Vancomycin-resistant E. faecalis. The peptide did not show any cytotoxic activity, while DLS and TEM studies demonstrated that it exists as nanoparticles in aqueous media. The zeta potential study confirmed the positive charge on 79, indicating higher stability and less aggregation.¹¹⁶

Figure 32.

Structure of the lipopeptide, all-hydrocarbon stapled peptide and the comparative structure of the short cationic antibacterial peptide.

A cationic peptide, KKKKKK-AAFAAWAAFAA-NH₂ (80, Figure 32) with separate hydrophobic and positively charged domains exhibited high antibacterial potency and low toxicity.¹¹⁷ To optimize the proteolytic stability and activity, 80 was cyclized to obtain peptide 81. The modified alanine residue, 2-(4’-pentenyl)alanine, was substituted in the peptide (80) at Ala¹⁰ and Ala¹⁴ to introduce an 8-carbon staple containing a helical structure. Peptide 81 was tested in vitro against E. coli (BL21) and hemolytic toxicity. The MICs were found in the range of 1-4 μM comparable with that of 80 (~2 μM). The parent peptide (80) was selective towards the bacterial membrane with an MHC value of >320 μM, while the stapled analog 81 showed increased toxicity with an MHC value of 3.8 μM.¹¹⁸

The human intestinal peptide transporter (hPEPT1) is the gateway receptor for the absorption of naturally occurring peptides. A study on the permeation of a library of short cationic antimicrobial peptides was initiated, and their interaction in hPEPT1 expressing Xenopus laevis oocytes was studied. Peptide RWR-NH-Bn (82, Figure 32) had an affinity for hPEPT1 and was the efficacious antimicrobial agent with a flux rate of 3.8 pmol/(s.cm²) and ~88% inhibition of the oocytes. The MIC values were found to be 75 and 25 μg/mL against S. aureus and MRSA, respectively, for 82. One of the most bioactive antimicrobial dipeptides, TbtR-CH₂-Bn (83) did not show hPEPT1 interaction due to its membrane disruptive action and was studied for the passive membrane transportation. The study was done using a phospholipid vesicle-based permeation assay (PVPA) model, and excellent absorption was observed for 83 with a P_app value of 5.27 (10^-6 cm/s) and enhanced antimicrobial activities (MIC = 2.5 μg/mL against S. aureus and MRSA and 10 μg/mL against E. coli). Hence, it was observed that even if the peptides do not act as the substrate for hPEPT1 they still possess enhanced activity and the potential to be orally active through passive permeation.⁷⁶

4.3.2. Antifungal agents

Antifungal peptides in general act via membrane disruption and pore formation, creating interference in cell wall synthesis. A few peptide drugs containing unnatural amino acids are being used in hospitals, such as caspofungin, rezafungin and anidulafungin. Herein, we have discussed a few key examples of the development of short peptides as antifungals, with a primary focus on the unnatural amino acids that played a key role. Modified side chain amino acids have been used to develop potent antifungal scaffolds through rationally designed sequences.

Membrane disruption:

Ghrelin, a 28-amino acid-containing antimicrobial peptide (AMP), demonstrated bactericidal activity against E. coli and P. aeruginosa.¹¹⁹ A truncated hexapeptide Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH₂ showed an IC₅₀ value of 6.8 μM against C. albicans and an MIC value of 6.8 μM against Cryptococcus neoformans. Modifications of this hexapeptide resulted in the elongated nonapeptide Arg-D-Trp-D-Phe-Ile-D-Phe-His-Lys-Lys-Arg-NH₂ (IC₅₀ = 1.64 μM, C. albicans).¹²⁰ A series of hexapeptides based on growth hormone-releasing peptide (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂) were developed that showed potent activity against C. albicans and C. neoformans. The key constituent amino acids were replaced with specific unnatural amino acids without altering the chain length, and the peptides Orn-D-Trp-Cha-Ile-D-Phe-His(1-Bzl)-NH₂ (84) and Orn-D-Trp-D-Phe-Phe(4-Me)-D-Phe-His(1-Bzl)-NH₂ (85) (Figure 33) showed good activities against C. albicans, MRSE, and MRSA.¹²¹ These two lead sequences were optimized by keeping hydrophilic amino acid residues in 84 and hydrophobic amino acid residues in 85 intact and replacing systematically the rest of the amino acids to alter the amphiphilicity and bulkiness.¹²² In peptide 84, replacing D-Phe⁴ with Cha gave peptide 86 (IC₅₀ = 0.46 μg/mL, MIC = MFC = 1.25 μg/mL), while in peptide 85, replacement of His(1-Bzl)⁶ by Orn and D-Lys gave peptides 87 (IC₅₀ = 0.46 μg/mL, MIC = MFC = 0.63 μg/mL) and 88 (IC₅₀ = 0.40 μg/mL, MIC = MFC = 0.63 μg/mL) against C. neoformans comparable to the standard antifungal drug, amphotericin B (IC₅₀ = 0.69 μg/mL, MIC = MFC = 1.25 μg/mL). The mechanistic studies using SEM and TEM demonstrated the effect of peptides on the fungal cell wall.¹²²

Figure 33.

Structure of the lead hexapeptides and their unnatural amino acid substituted analogs.

Rationally designed histidines were used to develop new classes of short antifungal peptides.^123–130 The presence of a negative charge on the outermost membrane of a microbe imparts it with an overall negative charge. Therefore, to facilitate the interaction of the designed peptides with the fungal membrane, an overall positive charge was introduced in the peptide sequence by incorporating modified histidines. A series of tripeptides was synthesized (Figure 34) using C-2 arylated histidines and arginine with a benzylamide group at the C-terminal. The in vitro activity revealed the best antifungal activity in peptides 89 and 90, with IC₅₀ values of 0.07 and 0.1 μg/mL and identical MIC and MFC of 0.16 μg/mL against C. neoformans. The mechanistic studies demonstrated the effect of positively charged peptides on the negatively charged cryptococcal cell membrane.¹³¹ Further, dipeptides such as His[2-(4-tert-butylphenyl)]-Arg-NHBzl (91) and Trp-His[2-(4-biphenyl)]-NHBzl (92) were identified that showed IC₅₀ values of 0.16 μg/mL and 0.54 μg/mL, respectively, against C. neoformans, which was 4 times higher than the standard drug amphotericin B. The peptides were found to be non-toxic up to 10 μg/mL. It was concluded that the peptide with suitable lipophilic derivatization of histidine demonstrated the most potent antifungal activity.¹²⁸

Figure 34.

Structure of unnatural amino acid containing antifungal peptides.

In continuation, the role of two unnatural histidine residues in the peptidic sequence was investigated with L-Trp as the central residue in the sequence. Lead His[2-(4-biphenyl)]-Trp-His[2-(4-biphenyl)]-OMe (93, Figure 34) was identified as having promising activity (IC₅₀ = 0.35 μg/mL and MIC = MFC = 0.63 μg/mL) against C. neoformans. Peptide 93 also showed good antibacterial activity (IC₅₀ = 1.93 μg/mL, MIC = 5 μg/mL) against MRSA. Peptide 93 also displayed synergistic action with fluconazole and amphotericin B against C. neoformans and was non-cytotoxic and non-hemolytic with high proteolytic stability. The mechanism of action study demonstrated that the peptide translocated into the cell and caused nuclear fragmentation along with cell membrane disruption. Peptide 93 was twice as potent as amphotericin B and displayed enhanced proteostability due to the incorporation of unnatural amino acids.¹³⁰ Subsequently, another series of tripeptides [His-Trp-Arg], was synthesized and explored for anticryptococcal activity. This study led to His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe (94, Figure 34) that showed anticryptococcal activity (MIC = 0.63 μg/mL and IC₅₀ = 0.37 μg/mL). The cytotoxicity assay in a non-cancerous mammalian cell line VERO suggested that 94 was non-toxic, and mechanistic studies confirmed its selective affinity towards the fungal cell membrane.¹³²

In the quest to discover novel and highly potent antifungal peptides, unnatural amino acids such as N-1-substituted histidines were explored. Tripeptide His[1-(4-n-butylbenzyl)]-Trp-His[1-(4-n-butylbenzyl)]-NHBn (95, Figure 34) showed MIC and IC₅₀ values of 2.46 and 1.36 μg/mL against C. neoformans.¹³³ Another antimicrobial dipeptide, Trp-His[1-(3,5-di-tert-butylbenzyl)]-NHBn (96) produced MIC and IC₅₀ values of 3.81 and 2.1 μg/mL against C. neoformans. Peptide 96, also produced identical MICs and IC₅₀s of 8 and 4.4 μg/mL against E. faecalis and S. aureus, respectively.¹³⁴ These peptides were non-cytotoxic, non-hemolytic and synergistic with amphotericin B. Mechanistic studies revealed fungal membrane disruption and pore-formation leading to shape-change of the cells and cell lysis.

4.3.3. Antiviral agents

Viruses possess a unique ability to mutate rapidly, so the development of therapeutic interventions for viral infections has been a formidable task. Several encounters with the outbreak of Ebola, Zika viruses and the Covid-19 pandemic have steered research towards development of antiviral agents. Herein, we highlighted the role of unnatural amino acids in the development of antiviral agents effective in influenza, AIDS, hepatitis, COVID-19, and Zika virus infections, primarily targeting viral proteases. The introduction of unnatural amino acids has not only improved the potency of the already existing agents but also improved their pharmacokinetic profiles.

4.3.3.2. Anti-HIV

RRE inhibitors:

HIV-1 Rev response element (RRE) is an RNA that regulates viral replication by interacting with its ligand Rev and is considered an attractive target to block HIV-1 replication. Several synthetic ligands, including small molecules, peptides, peptidomimetics, and nucleotides, have been evaluated to disrupt the RRE-Rev interactions, but none were successful due to limitations pertaining to RNA surface area, conformation, and interaction with the cognate ligand. A unique 46,656-member peptide library containing unnatural amino acids was screened to find hits against RRE IIB RNA. Later, branched peptides were developed by incorporating two unnatural amino acids containing boronic acid to enhance the proteolytic stability along with selectivity and binding affinity towards the primary Rev binding site RRE IIB of HIV-1 RNA. The dissociation constant (K_d) was estimated for the selected 22 peptides obtained from high-throughput screening. Peptides demonstrating submicromolar K_d were evaluated in vitro against RRE IIB. The branched peptide (GGD)₂PGLY (97, Figure 35) exhibited 80% inhibition of Rev-RRE and inhibited HIV-1 p24 production with an IC₅₀ value of 15.4 μM and a cytotoxicity concentration CC₅₀ of 135 μM. The biochemical investigations were performed using RNase protection and SHAPE-MaP assays, which demonstrated the binding sites of the 97 to be Rev proteins. The unnatural amino acid containing branched 97 was proven to bind efficiently to the RNA and change its conformation to compete with the Rev protein.¹³⁵

Figure 35.

Structure of the branched peptide efficiently binds to the Rev binding site of the HIV-1 RNA.

HIV-1 protease inhibitor:

Atazanavir (ATV, 98, Figure 36) is an azapeptide and efficacious HIV-1 protease inhibitor (PI), but it possesses poor solubility and absorption. In an attempt to improve the PK profile of ATV, a series of phosphate and amino acid ester-based prodrugs was developed. The synthesized prodrugs were evaluated for solubility and stability over a range of pHs and in vivo PK profiling. The ATV phosphate ester failed to fully convert into the parent drug and could not achieve the effective systemic level of the ATV, whereas amino acid ester prodrugs improved the PK profile of the ATV. The best results were observed with the L-Phe-Sar ester prodrug (99), showing 4 times more exposure (AUC) and 8 times better C_24h (concentration achieved after 24 h of oral administration) as compared to ATV itself at a similar dose. The sarcosine (Sar) linker between the L-Phe and ATV facilitates enhanced drug release by cyclization of the pro-moiety (L-Phe-Sar) at neutral pH, as compared to the L-Val prodrug of the ATV (100). The role of sarcosine was demonstrated by the comparative analysis of the relative ATV AUC of the prodrugs 99 and 100, which was 399% and 11%, respectively.¹³⁶ With the aim of further improving the pharmacokinetic profile of the ATV, several prodrugs were developed by incorporating N,N-dimethyl-L-Val and N,N-dimethyl-D-Val. The chemical stability improved with the L-Val prodrug and was further taken for linker optimization. The (carbonyl)oxyethyl-N,N-dimethyl-L-valinate isomer (101) was found to demonstrate the best results with a minimum t_1/2 of 215 h at different pH values and 500-fold enhanced stability in aqueous media. The in vivo PK studies after oral administration showed C_max (maximum concentration) and C₂₄ of 2700 nM and 784 nM, respectively, and t_max of 0.4 h and AUC of 33421 nM.h. The in vitro profiling of the prodrug demonstrated plasma stability with t_1/2 ~25 min and FaSSIF (fasted state simulated intestinal fluid) solubility of 11 μg/mL.¹³⁷

Figure 36.

Strategies to obtain amino acid ester prodrugs of Atazanavir.

4.3.3.5. Anti-corona Agents

Cysteine protease inhibitors:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the major causative agent of the deadly coronavirus disease 2019 (COVID-19) pandemic outbreak. Cysteine protease M^pro is involved in the active cycle of the coronavirus and has emerged as an attractive antiviral target. M^pro inhibitors were designed using in silico analysis of the protease binding of substrates synthesized via a covalent bond between the thiol group of cysteine and the aldehyde group of inhibitors. To enhance the binding of the inhibitor to different pockets of the protease, ring systems such as cyclohexyl or 3-fluorophenyl were incorporated. The incorporation of unnatural amino acids, i.e., D-cyclohexylalanine and D-Phe(4-F), enhanced the binding to the S2 binding pocket of the SARS-CoVM^pro. The designed analogs 102 and 103 (Figure 37) demonstrated 100% and 96% inhibition of SARS-CoVM^pro at 1 μM. Fluorescence resonance energy transfer (FRET)-based cleavage assay of 102 and 103 showed IC₅₀s of 0.053 and 0.04 μM, respectively. The crystal structure analysis provided the inhibitory mechanism of 102 and 103, which showed the interaction of the aldehyde group, the (S)-γ-lactam ring, and the amide bonds with the active sites of the M^pro. The EC₅₀ values were observed to be 0.53 and 0.72 μM for 102 and 103, whereas the CC₅₀ value was >100 μM. The druggability was evaluated in vivo, and a good pharmacokinetic profile of 102 was revealed with a t_1/2 of ~4 h, a bioavailability of 87.8%, and no signs of toxicity.¹³⁸ With the aim of developing broad-spectrum antiviral protease inhibitors, a known antiviral, rupintrivir (AG7088) (EC₅₀ = 0.009 μM), was investigated against enterovirus 71 (EV71) 3C^pro that exhibits a poor pharmacokinetic profile and no activity against SARS-CoV 3CL protease. A peptidomimetic antiviral inhibitor was designed by combining the active pharmacophoric fragments, such as the (S)-γ-lactam ring, and the phenyl/heterocyclic rings. From the designed series, the analog containing the indole ring and an aldehyde group (104, Figure 37) interacted effectively with the EV71 3C^pro (IC₅₀ = 2.36 μM), confirmed by the in silico crystal structure analysis. The broad-spectrum antiviral activity of 104 was evaluated against various strains of enterovirus and rhinovirus and demonstrated EC₅₀s in the range of 0.03-4 μM. Analog 104, when tested against SARS-CoV-2, showed an IC₅₀ of 0.034 μM against 3CL^pro (EC₅₀ = 0.29 μM, CC₅₀ = 808.7 μM). The pharmacokinetic profile of 104 revealed a longer t_1/2 of 5.85 h and a bioavailability of >100%. Conclusively, 104 showed the potential to become a broad-spectrum antiviral, including an antiCorona virus agent.¹³⁹

Figure 37.

Structures of the SARS-CoV-2 inhibitors.

Sec61 inhibitors:

Sec61, a translocon essential for viral proteostasis, is an attractive target for the apratoxins that could combat the SARS-CoV-2 and other viral infections. Apratoxin S4 (Apra S4, 105, Figure 37), being an antiviral and an anticancer compound, was evaluated against SARS-CoV-2. Upon evaluation in vitro for antiviral activity, 105 produced IC₅₀s of 0.17 and 0.71 μM against SARS-CoV-2 infected Vero E6 and HeLa-hACE2 cells, and significantly decreased viral replication with no cytotoxicity was observed for the 105 at 10 μM in a human cell model. Moreover, 105 demonstrated antiviral activity with IC₅₀s of 15, 14, 3.3, and 0.46 nM against Zika, West Nile, Dengue, and Influenza A viruses, respectively. The validation of the Sec61-mediated effect was done by using the Sec61 inhibitor, eeyarestatin I, and siRNA-induced knockdown of Sec61A1. The mechanism of action of 105 was deciphered by microscopic studies, confirming its role in affecting viral proteostasis and replication organelles by targeting the translocon complex. It was also observed to reduce ACE2 protein production at 1 μM. Analog 105, a synthetic hybrid with a cyclic structure containing unnatural amino acids, is a broad-spectrum antiviral effective against an array of viral infections.¹⁴⁰

4.4. Unnatural amino acids containing agents for cardiovascular diseases

Cardiovascular (CV) function is largely regulated by hormones and ligands which include urotensins, melanocortins, catecholamines, natriuretic peptides, and many more. Most of these agents are peptidic in nature; therefore, natural and modified peptides and amino acids have played a huge role in CV drug discovery. Pharmacological optimization of molecules by utilizing unnatural amino acids has led to the development of various peptide-based therapeutic agents for CV disorders and is discussed in this section.

Urotensin Receptor Blockers:

The urotensinergic system is comprised of two endogenous ligands, namely, urotensin II (hUII) and urotensin II-related peptide (URP, 106, Figure 38), acting through the urotensin receptor (UT). Both ligands possess a similar hexapeptide cyclic core, differing only at the N-terminal, act as potent vasoconstrictors, and have grabbed attention for their role in maintaining cardiovascular homeostasis. Two potent URP analogs viz. (Bip⁴)URP (Urocontrin C) and (Pep⁴)URP (Urocontrin A), were developed as UT allosteric modulators by replacing the Trp⁴ residue of URP with unnatural amino acids.¹⁴¹ Later, modification of URP was carried out to obtain analogs that could interact with the urocontrin allosteric cavity efficiently. The effect of spatial orientation, size, and spacing between the aromatic moieties in the cyclic core of URP was explored by utilizing unnatural amino acids. Two analogs [D-Bip⁴]URP (107) and [D-Pep⁴]URP (108) along with their synthetic precursor [D-Phe(4-I)⁴]URP (109) were identified. These D-analogs exhibited similar binding affinity to that of URP (pIC₅₀ = 8.29), whereas their L-counterparts showed 5 to 31 times lower binding affinity. On further evaluation for aortic ring contraction, it was observed that analogs 107 (pEC₅₀ = 7.32), 108 (pEC₅₀ = 7.55), and 109 (pEC₅₀ = 7.54) act as agonists with less potency than URP (pEC₅₀ = 8.16). The L-counterpart of 107, [Bip⁴]URP (pEC₅₀ = 6.47)², showed very weak agonism, and [Pep⁴]URP was completely devoid of agonist activity. Two new UT peptidic allosteric modulators, [Trip⁴]URP (110) and [Phe(4-Py)⁴URP] (111), were effective in blocking hUII-induced contraction without altering URP vasocontraction. Both analogs depict a strong binding affinity for UT, i.e., pIC_50s of 6.87 and 6.81, and exert a potent antagonistic effect by reducing hUII-induced contraction to 79% and 70% of E_max at 10^-6 M concentration. These analogs with UT allosteric modulator effect could be useful in the treatment of cardiovascular disorders.¹⁴²

Figure 38.

UT non-competitive antagonists.

Apelin receptor agonists:

Apelin-13 (112, Figure 39), a ligand to the apelin receptor (APJ), regulates blood pressure and cardiovascular functions by decreasing the mean arterial pressure (MAP) and exerting hypotensive and positive ionotropic effects. APJ is one of the emerging therapeutic targets for the treatment of cardiovascular diseases.¹⁴³ In the quest to discover a stable and potent ligand of APJ, apelin-13 analogs were generated by incorporating unnatural amino acids at the 13^th position. The Phe¹³ residue of apelin-13 was replaced by aromatic amino acids, which resulted in a significant increase in binding affinity and improved potency, suggesting the critical existence of π-π interactions between the C-terminus of the ligand and the APJ receptor. Tyr(OBn)¹³ derivative, Pyr-RPRLSHKGP-Nle-P-Tyr(OBn) (113), is the most potent APJ ligand reported till now that exhibits a 60-fold increase in binding affinity (IC₅₀ = 0.02 nM and K_i = 0.016 nM) when compared with the native peptide 112. Similarly, replacement of Phe¹³ by sterically hindered Bpa resulted in peptide 114 (Pyr-RPRLSHKGP-Nle-P-Bpa), while replacement of Phe¹³ with (α-Me)Phe gave 115 (Pyr-RPRLSHKGP-Nle-P-(α-Me)Phe). Both peptides displayed a 30-fold improvement in cAMP accumulation inhibition compared to the native peptide, with EC_50s of 0.04 nM and 0.07 nM, respectively. Peptide 113 depicted strong β-arrestin-1 recruitment at 7.4 nM and Gα_oA subunit activation with an EC₅₀ of 1.4 nM. In vivo, 115 displayed a sustained decrease in MAP compared to 112 upon iv administration at doses of 0.1 to 1 mg/kg in Sprague-Dawley rats, depicting the strong hypotensive effect of the peptide. Peptide 113 was found to be stable with a superior half-life of 1 h in rat plasma compared to apelin-13, highlighting the importance of unnatural amino acid-mediated C-terminal modification of 112 for the discovery of ligands for APJ.¹⁴⁴

Figure 39.

Apelin-13 analogs.

Melanocortin receptor antagonists:

A series of potent melanocortin ligands were identified by a double simultaneous replacement strategy within the previously reported peptide Ac-His¹-DPhe²-Arg³-Trp⁴-NH₂ (116, Figure 40). Diversification of 116 was carried out by replacing amino acids at positions 1, 2, or 4 with natural and unnatural amino acids, along with the addition of octanoyl group at the N-terminus. Simultaneous substitution at position 4 with Bip and the octanoyl group capping at the N-terminus gave octanoyl-His-DPhe-Arg-Bip-NH₂ (117), which exhibited antagonistic potential at mMC4R with a pA₂ value of 7.0 and stimulatory activity of 50% when assayed up to 100 μM at mMC3R. Concurrent substitution at position 1 with amino acids and position 2 with D-Phe(4-I) led to analogs with antagonistic potential at mMC3R and mMC4R. The tetrapeptide Ac-4-Pal-D-Phe(4-I)-Arg-Trp-NH₂ (118) was most potent toward mMC4R with 66-fold selectivity compared to mMC3R with pA₂ values of 8.9 and 7.1, respectively. The presence of D-Phe(4-I) at different positions of the melanocortin template showed antagonist activity, the use of a double simultaneous replacement approach reversed the activity pattern. For example, peptide 119 with D-Phe(4-I)² and Tic⁴ combination exhibited full agonistic activity with EC₅₀s of 2.9 μM and 0.64 μM at mMC3R and mMC4R, respectively. Thus, double simultaneous substitution modulates the potency, selectivity, and pharmacology of the peptide and could be utilized for further development of leads for melanocortin receptors.¹⁴⁵

Figure 40.

Melanocortin receptor antagonists.

4.5. Unnatural amino acids containing agents for metabolic disorders

Metabolic disorders include hyper- and hypo-glycemia, obesity, pancreatic, and kidney diseases. The etiology of most of these disorders is based on hormonal dysregulation; hence, peptide-based agents, which mimic these hormones or act as ligands to the biological pathways, have become the most therapeutically relevant class of candidates for drug discovery. Insertion of unnatural amino acids in naturally occurring peptides like glucagon, thyroid hormone progenitor T4, melanocortin, and peptide YY has proven to show promising activities in metabolic disorders.

4.5.1. Hyper/Hypoglycemia

PPARγ agonists:

Peroxisome proliferator-activated receptor gamma (PPARγ) is a therapeutic target in several conditions, including diabetes mellitus. A study about the activity of thyroid hormone (TH) progenitor T4 (120, Figure 41) and its oxidative deaminated metabolites, 3,3′,5,5′-tetraiodothyroacetic acid (TETRAC, 121) and 3,3′,5-triiodothyroacetic acid (TRIAC, 122) as potential agonists of PPARγ and retinoid X receptor (RXR) was reported. TETRAC (177) exhibited significant PPARγ activation (K_d = 0.11 μM, EC₅₀ = 0.10 μM), higher than pioglitazone. A similar agonistic potential was observed on RXRα (K_d = 1.5 μM, EC₅₀ = 1.32 μM). The coactivator recruitment assay displayed a higher potency of 121 than rosiglitazone towards PPARγ for promoting steroid receptor coactivator 1 (SRC-1). Analogs 120 and 121 induced PPARγ-regulated gene expressions, marked by upregulation of CD36 protein in murine 3T3-L1 fibroblasts and human liver cells (HepG2). The pharmacokinetic observation revealed that TH-induced PPAR signaling effect was likely due to endogenously formed 121 from 120. The mice treated with TH mimetic Br-TETRAC (123) (10 mg/kg, orally) produced 21-fold increase in PPARγ activity compared to 120. The X-ray crystal structure of the TETRAC-PPARγ complex displayed unique bipolar binding interactions between 121 and the PPARγ ligand binding site. Presence of binding interactions of 121 with Tyr327, His449, and Lys367 in basic region and Arg288, Ser342, and Glu343 of the PPARγ provide stability to the TETRAC-PPARγ complex. The simultaneous activation of PPARγ and RXRα in heterodimers using the TH chemotype template potentiates PPAR signaling and is a potential strategy for PPAR-targeted therapy.¹⁴⁶

Figure 41.

THs and TH-mimetics as PPARγ/RXRα agonists.

Glucagon receptor agonists:

Glucagon (124, Figure 42) is an endogenous hormone that regulates the level of glucose by binding to the glucagon receptor (GCGR) and increasing the glucose concentration in the blood via gluconeogenesis and glycogenolysis. Its application as a therapeutic agent for the treatment of severe hypoglycemia is curtailed due to its short half-life. Synthesis of a series of backbone modified glucagon analogs was carried out using α/sulfono-γ-AA residues and other unnatural amino acids. A protraction strategy was utilized to add a long fatty acid chain to the peptides. Peptide 125 was developed by replacing the methionine (Met) and asparagine (Asn) residues of glucagon with one sulfono-γ-AA residue (X¹), which showed potent activity (EC₅₀ = 0.28 nM) compared to control O8 (EC₅₀ = 0.46 nM) in the cAMP response element (Cre)-luciferase assay. The inclusion of two to five sulfono-γ-AAs in the sequence of glucagon results in peptides devoid of activity. Later, Aib was introduced in the sequence by replacing serine to obtain peptide 126, which showed potent activity (EC₅₀ = 0.86 nM). A chain of fatty acids was introduced at different positions of 126 in order to improve PK properties. Peptide 127, possessing a fatty acid chain at K17, retained the activity. This analog was also evaluated in the cAMP assay and exhibited results comparable to the Cre-Luc assay. The serum stability study of 127 showed no degradation in RP-HPLC after 24 h whereas glucagon was degraded by 85%. In vivo activity demonstrated an improved metabolic stability profile for 126 with a half-life (4 h), while glucagon was metabolized within 15 min. The ability to increase the blood glucose level was determined by an intraperitoneal insulin tolerance test, where female mice with a high dose of glucagon showed a higher blood glucose level, whereas 127 exhibited a lower potency of glucagon.¹⁴⁷

Figure 42.

Unnatural amino acid and sulfono-γ-AA residues containing glucagon analogs.

4.5.2. Obesity

Melanocortin receptor antagonists:

Cyclic peptides were identified as potent and selective ligands of melanocortin receptor 4 (MC4R), a target of interest in metabolic disorders such as obesity, feeding regulation, and sexual dysfunctions. On the basis of crystal structure of hMC4R complexed with the non-selective MC3/MC4 receptor antagonist, SHU-9119 (128, Ac-Nle⁴-c[Asp⁵-His⁶-D-Nal(2’)⁷-Arg⁸-Trp⁹-Lys¹⁰]-NH₂), hydrophobic spaces in the receptor pocket that interact with the side chain of amino acid 7 were identified (Figure 43). Recently approved cyclic nonselective melanocortin agonist MT-II (129) was used as standard, and it was observed that replacement of D-Phe⁷ with D-Phe(mNp(1’)) resulted in compound SBL-MC-49 (130), which possessed full-agonistic activity on MC4R (pEC₅₀ = 7.5 nM) and efficacy of 107% and was a partial agonist (pEC₅₀ = 6.81 nM) and efficacy around 95%. At the same time, incorporation of unnatural D-Orn residue at position 4 of 128 resulted in SBL-MC-37 (131), which showed 100-fold selectivity with inhibitory potency (pEC₅₀ = 7.3 nM) at the MC4R over the MC3R, while L-Orn substitution at the same position resulted in 21-fold change in selectivity of the ligand for the MC4R over the MC3R. Several other replacements with unnatural amino acids, such as Trp(5-Cl)⁹ and Trp(7-F)⁹, led to improved selectivity at the hMC4R. Thus, the melanocortin receptor subtype specificity of the ligands can be achieved by employing such strategies in order to fully understand the therapeutic potential of these receptors.¹⁴⁸

Figure 43.

Selective melanocortin receptor subtype cyclic peptides.

Linear tetrapeptides based upon the melanocortin peptide Ac-His-D-Phe-Arg-Trp-NH₂ (132, Figure 43) were identified as potent MC4R agonists. The peptide-backbone-based modification approach, through the incorporation of β³-amino acids at different positions of the sequence, gave hetero-tetrapeptides. Amongst analogs, β³-hTrp⁴ containing peptide 133 produced 35-fold selectivity at mMC4R compared to mMC3R and agonist potencies of 12 nM at mMC4R and 415 nM at mMC3R. Such bioactive compounds can act as templates for MC4R-selective agonists and have utility in the development of efficient MC4R inhibitors.¹⁴⁹

These examples of UAAs-based peptides showed that therapeutic development of peptide-based drugs can be successfully achieved, and peptides can be chemically optimized for the desired therapeutic profile. The optimization of one parameter can lead to unfavorable changes in other properties such as bioactivity, affinity, and target selectivity. However, the careful design of UAAs and their strategic placement can lead to the identification of peptide drugs. Optimizing the pharmacological profile of peptides is more challenging than small-molecule compounds due to the complex nature of peptides and the limited number of chemical tools available to optimize them. The UAAs-based approach is very promising for tailoring the physicochemical and pharmacological profiles of peptides. The required chemical changes depend on the disease targets vs. necessary changes vs. target pharmacological profile. Therefore, researchers need to carefully evaluate the desired therapeutic profile, study the disease targets, and then design their peptide optimization approach. There are no generalized patterns for improving the drug-like properties of peptides, but there are several chemical tools and approaches that can be employed to achieve the desired profile and clinical agents.

5. Conclusion and future perspective

Since the introduction of liothyronine in 1956, the utility of unnatural and modified amino acids has been explored in drug discovery. The increased commercial availability of unnatural amino acids led to the discovery of several biologically active ligands. As of today, unnatural and modified amino acids are components of more than 110 FDA-approved drugs with application in a variety of metabolic disorders, infectious and cardiovascular diseases, analgesics, and oncology. As evident from the examples across several disease domains, amino acid replacement or modification in peptides results in the improvement of multiple parameters, including potency, stability, efficacy, reduced toxicity, target selectivity and specificity. The unnatural and modified amino acids, therefore, serve as vital building blocks for constructing biologically relevant scaffolds. Different classes of amino acids, such as β, γ, and δ, along with non-proteogenic side chains containing amino acids, have played an essential part in the design and synthesis of pseudopeptides, peptide mimetics, and small molecules. The application of unnatural amino acids in the design and synthesis of peptides plays a vital role in the identification of new target molecules with pharmacological profiles and the optimization of various ADME/PK parameters. Larger pools of unnatural amino acids resulted in more peptide- or peptide-type drugs coming through the rigors of clinical trials, with some even surviving oral dosing. One assumes that this capability could expand through the emergence of even larger pools of commercially available unnatural amino acids to facilitate drug discovery campaigns.

The introduction of non-coded building blocks also holds immense promise when modifying natural peptides and proteins to improve physicochemical properties and desired pharmacological profile. UAAs-based approaches are also useful for optimizing the solubility and membrane permeability, potency, and selectivity of drugs, therefore improving the biodistribution and pharmacodynamics of peptides. Although there are no general patterns for modulating the pharmacological properties of peptides, researchers still need to assess the biological targets, metabolic sites, peptidases, and nature of pharmacophore motifs to carry out desired chemical changes through UAAs. Ultimately, chemical optimization through employment of UAAs in conjunction with other existing approaches like cyclization is highly useful for hit-to-lead optimization to deliver clinically useful compounds.

This perspective provides a resource for researchers to appreciate the impact that unnatural amino acids and UAAs-based approaches have on peptide- and small-molecule drug discovery. This area of research has undergone rapid transformation during the past three decades, exemplified by the success of many clinically approved drugs. It can be confidently predicted and anticipated that the number of clinically approved unnatural amino acid-based drugs to treat multiple diseases will only increase in the years ahead.

Highlights.

• UAAs are widely present in clinical agents, and >110 drugs are in clinical use

• 44% of FDA-approved UAAs containing drugs are have orally available routes of administration

• UAAs play a critical role in improving the “drug-likeness” of peptides

• Side chain modified UAAs are vital tools for optimization of multiple pharmacological parameters (i.e. in-vivo stability, cell permeability, bioavailability, and oral absorption)

• Block metabolic sites and improve lipophilic vs hydrophilic ratio and deliver better drug-like profiles)

• N-methylated UAAs are reliable modifications for improving permeability

• UAA-based approaches allow researchers to explore peptides and peptidomimetic molecules against various diseases such as cancer, metabolic disorders, infections, cardiovascular neurological issues ect

Statement of Significance.

• This Perspective articulates the significant role that unnatural amino acids play as structural components and design elements of U.S. FDA-approved drugs.

• This study reveals a (i) systematic analysis of unnatural amino acids contained in FDA-approved drugs, (ii) the role in tuning physicochemical properties, and (iii) the diverse unnatural amino acids as critical tools in various disease areas and their role in tuning pharmacological profiles.

• Future directions for unnatural amino acids in drug discovery are described.

ABBREVIATIONS USED

ADC

antibody-drug conjugates

ADME

absorption, distribution, metabolism and excretion

AMP

antimicrobial peptide

ATV

atazanavir

AUC

area under the curve

BBB

blood brain barrier

cAMP

cyclic adenosine monophosphate

CHO

chinese hamster ovary

CNS

central nervous system

DELs

DNA-encoded libraries

DLS

dynamic light scattering

DOR

δ-opioid receptor

ERK

extracellular signal-regulated kinase

FDA

US food and drug administration

FR

folate receptors

GABA-AT

γ-aminobutyric acid aminotransferase

GCGR

glucagon receptor

GPI

Guinea pig ileum

GTP

guanosine triphosphate

HIV

human immunodeficiency virus

hPEPT

human intestinal peptide transporter

hPEPT1

human intestinal di- and tripeptide transporter 1

HPLC

high performance liquid chromatography

HTRF

homogeneous time resolved fluorescence

IC₅₀

50% inhibitory concentration

IHC

immunohistochemistry

KOR

κ-opioid receptors

LAA

lipoamino acids

MAP

mean arterial pressure

MFC

minimum fungicidal concentration

MIC

minimum inhibitory concentration

MOR

μ₁-opioid receptor

MRSA

methicillin-resistant Staphylococcus aureus

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

MVD

mouse vas deferns

NADPH

nicotinamide adenine dinucleotide phosphate

NRP

nonribosomal peptides

NT

neurotensin

PAMPA

parallel artificial membrane permeability assay

PCFT

proton-coupled folate transporter

PI

protease inhibitor

PK

pharmacokinetic

PPARγ

peroxisome proliferator-activated receptor gamma

PVPA

phospholipid vesicle-based permeation assay

qPCR

quantitative polymerase chain reaction

RFC

reduced folate carrier

RRE

rev response element

RXR

retinoid X receptor

SAR

structure activity relationship

SARS-CoV-2

severe acute respiratory syndrome corona virus 2

SEM

scanning electron microscopy

STAT

signal transducer and activator of transcription

TEM

transmission electron microscopy

TH

thyroid hormone

ThT

thioflavin T

UAA

unnatural amino acid

URP

urotensin II-related peptide

UT

urotensin receptor

VRE

vancomycin-resistant enterococcus

VRSA

vancomycin-resistant Staphylococcus aureus

Biographies

Krishna K. Sharma received MS (Pharm.) and PhD degrees from NIPER, Mohali, India, under the supervision of Rahul Jain and worked on peptides therapeutics. He did postdoctoral research at the School of Pharmacy at the University of Kansas, Lawrence, USA and the College of Pharmacy at the Ohio State University, Columbus, USA. He works as a Senior Research Scientist at the Department of Chemistry at Iowa State University in Ames, USA. His research interests include synthetic organic chemistry for peptide modifications and peptide-based therapeutics in various disease areas.

Komal Sharma received her MS (Pharm.) from NIPER, Mohali, India, in carbohydrate chemistry. She obtained her PhD under Rahul Jain and worked on the synthesis, biological, and mechanistic investigation of short antimicrobial peptides. Her research interests include the design and development of peptide-based therapeutics and biomaterial chemistry. She is now pursuing her passion for research at Stanford University.

Kamya Rao obtained her MS (Pharm.) from NIPER, Ahmedabad, in 2021. She is pursuing a Ph.D. under the guidance of Rahul Jain and conducting research on the synthetic modifications of amino acids and their utilization in biologically active peptides.

Anku Sharma, after earning an earning an MS (Pharm.) from NIPER, Hyderabad, moved to Jamia Hamdard, New Delhi, and worked as a Junior Research Fellow in a DST-BRICS project with Ahmed Kamal. He is currently pursuing a PhD under the guidance of Rahul Jain. His research interests include the modification of biologically important amino acids and peptides and studying their mechanisms of action.

Gajanan K. Rathod obtained a PhD in medicinal chemistry under the guidance of Rahul Jain. and earlier completed a MS (Pharm.) from NIPER, Mohali. His current research area includes the development of strategies for the synthesis of pseudopeptides and modified amino acids. He also worked on the synthesis and evaluation of the 8-aminoquinolines for their antimalarial activities.

Shams Aaghaz completed an MS (Pharm.) from NIPER, Hyderabad, and then obtained a PhD with Rahul Jain. His research area includes the design, synthesis, biological evaluation, and mechanistic studies of peptides containing functionalized amino acids as potential antimicrobial agents. He is currently working as a postdoctoral associate at Baylor University, TX, USA.

Naina Sehra received an MSc in Chemistry from Visvesvaraya National Institute of Technology and then joined Rahul Jain for a PhD. Her research interests include the design and synthesis of potential amyloid beta inhibitors for the treatment of Alzheimer’s disease.

Rajesh Parmar is a MS (Pharm.) from NIPER Mohali and received a PhD working with Rahul Jain. His research interests include the design, synthesis, and biological evaluation of the anti-amyloid-β peptides as anti-Alzheimer’s agents.

Brett VanVeller is an Associate Professor in the Department of Chemistry at Iowa State University. He received his PhD from the Massachusetts Institute of Technology under the supervision of Prof. Timothy M. Swager. He later completed postdoctoral studies as a Canadian Institutes of Health Research Fellow under Prof. Ronald T. Raines, then at the University of Wisconsin–Madison. His research interests focus on physical organic chemistry, synthetic and bioorganic chemistry, and peptide therapeutics.

Rahul Jain moved to UT Southwestern Medical Center after a PhD in medicinal chemistry at CDRI, Lucknow, India. He returned to India after working at the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institutes of Health, and Tulane University, and joined NIPER, where he is professor and head of medicinal chemistry. His research interests include peptide/mimetic-based CNS and antimicrobial agents, new structural classes of synthetic amino acids, antimalarials, and antituberculosis agents.