Instructive Advances in Chemical Microbiology Inspired by Nature’s Diverse Inventory of Molecules

Abstract

Natural product antibiotics have played an essential role in the treatment of bacterial infection in addition to serving as useful tools to explore the intricate biology of bacteria. Our current arsenal of antibiotics operate through the inhibition of well-defined bacterial targets critical for replication and growth. Pathogenic bacteria effectively utilize a diversity of mechanisms that lead to acquired resistance and/or innate tolerance toward antibiotic therapies, which can result in devastating consequences to human life. Several research groups have established innovative programs that work at the chemistry—biology interface to develop new molecules that aim to define and address concerns related to antibiotic resistance and tolerance. In this Review, we present recent progress by select research groups that highlight a diversity of integrated chemical biology and medicinal chemistry approaches aimed at the development and utilization of chemical tools that have led to promising new microbiological insights that may lead to significant clinical advances regarding the treatment of pathogenic bacteria.

Several of Nature’s antibiotics have been effectively harnessed for the treatment of bacterial infections (see Figure 1).^1—3 Antibiotic agents that have entered the clinic contain exquisite structural features that enable selective inhibition of their corresponding bacterial targets. For instance, β-lactam antibiotics operate through a mechanism that relies on a strained electrophilic β-lactam warhead to covalently modify the nucleophilic hydroxyl of a key serine residue and inhibit a penicillin-binding protein involved in cell wall biosynthesis (Figure 2A, penicillin G 1).⁴ Tetracycline antibiotics bear an impressive molecular architecture that utilizes functionality that spans the entire ring system to form an intricate hydrogen bonding network and coordination to a magnesium(II) cation to inhibit bacterial ribosomes and prevent protein synthesis (Figure 2B, tetracycline 5).⁵ Natural product antibiotics not only have provided cures for those battling infection but also have led to critical insights regarding the biology of bacteria through extensive investigations at the chemistry—microbiology interface.^4–8

Figure 1.

Natural product antibiotics and their corresponding bacterial targets.

Figure 2.

Mode of action for (A) penicillin G (β-lactam), (B) tetracycline, and (C) streptomycin (aminoglycoside antibiotics).

Despite the significant advances that antibiotics and their synthetic derivatives have offered humans for nearly a century, bacteria utilize a series of mechanisms and phenotypic switches that enable them to evade antibiotic therapies during treatment (e.g., antibiotic resistance^1,9–15 and tolerance^{7,1 — 19}). It is important to note that our entire arsenal of antibiotic therapies was initially discovered as active growth inhibitors against rapidly dividing cultures of bacteria.^7,20 Conventional antibiotic therapies effectively target actively dividing bacterial cultures and operate through the inhibition, or disruption, of (1) cell wall biosynthesis,^21,22 (2) protein synthesis,^{23 – 26} (3) DNA synthesis,^27–30 (4) RNA polymerase,^31–33 (5) cellular membranes,^34,35 and (6) folate biosynthesis.³⁶

Actively replicating planktonic (free-floating) bacteria utilize one, or more, resistance mechanisms in a direct response to selective pressures experienced during antibiotic treatment (Figure 3).^37–40 Several distinct resistance mechanisms have been well-characterized in pathogenic bacteria, including: (l) mutation and modification to a target’s binding site^41–43 (e.g., penicillin binding protein mutations to mitigate the effects of penicillin), (2) enzymatic inactivation of antibiotics³⁷ (e.g., modification of the hydroxyl/amine groups of aminoglycoside antibiotics critical for binding to bacterial ribosomes via hydrogen bonding interactions), (3) utilization of efflux pumps to eliminate antibiotics that have entered bacterial cells before they inhibit their corresponding intracellular targets^44,45 (e.g., efflux of macrolide antibiotics), (4) Gram-negative bacteria utilizing their outer and inner membrane to prevent antibiotic entry and further reducing penetration through mutation,⁴⁶ (5) horizontal transfer of mobile genetic elements containing antibiotic-resistant genes⁴⁷ (e.g., plasmids), and (6) overproduction of bacterial targets to bypass the effects of antibiotics³⁷ (e.g., trimethoprim). Antibiotic-resistant bacteria are a significant threat to human health as they are present in nearly two million infections each year, many of which are life threatening (i.e., 23,000 deaths in the United States;³⁷ in 2015, there were 33,000 deaths in Europe from antibiotic-resistant infections⁴⁸).

Figure 3.

Comparison of acquired antibiotic resistance (planktonic cells) and innate antibiotic tolerance (bacterial biofilms).

In contrast to free-floating planktonic cells, bacteria are known to form surface-attached biofilm communities containing slow- or nonreplicating cells embedded within an extracellular polymeric matrix (Figure 3).^49–53 Bacterial biofilms have enriched populations of metabolically dormant persister cells that enable them to thrive under harsh environmental conditions that include host immune responses and aggressive antibiotic treatment regimes.^54–58 Due to their significantly altered metabolism, growth, and physiology compared to planktonic bacteria, biofilms are innately tolerant to all classes of antibiotics.⁷‘²⁰‘⁵⁹ Biofilms are the underlying cause of chronic and recurring infections and result in >500,000 deaths each year.⁶⁰‘⁶¹ Bacterial biofilms are known to be prevalent in the following infection, disease, and patient types: hospital-acquired infections (i.e., Staphylococcal infections), implanted medical devices (i.e., prosthetic joint, heart valve), catheter infections (i.e., central venous catheters, cerebrospinal fluid shunts), caries (tooth decay), cystic fibrosis (chronic lung infection), immunocompromised patients, skin/burn wounds, endocarditis, and osteomyelitis.⁶²

This Review highlights several research programs that are investigating a diversity of natural products as starting points to combat antibiotic-resistant and -tolerant pathogenic bacteria. These exciting programs not only showcase new molecules of interest but also offer insights regarding unique antibacterial strategies and efforts at the chemistry—microbiology interface to address problems related to pathogenic bacteria. The progress related to the programs detailed in this Review could provide ground-breaking clinical advances in the treatment of antibiotic- resistant and -tolerant infections.

ARYLOMYCINS: NEW GRAM-NEGATIVE ANTIBIOTICS THAT TARGET LepB

Arylomycin antibiotics are macrocyclic lipopeptides that inhibit bacterial type I signal peptidase (SPase), ^3,64 an essential membrane-bound protease that cleaves signal sequences from preproteins following their translocation across the cytoplasmic membrane utilizing a serine-lysine dyad^65,66 (e.g., arylomycin A-C₁₆ 12, Figure 4). There is considerable interest in advancing arylomycin antibiotics and related analogues for therapeutic applications. Baran and co-workers recently reported a scalable synthesis of arylomycins using an innovative C—H functionalization approach.⁶⁷ This section will highlight incredible work reported by Smith et al.⁶⁶ at Genentech regarding the development of a novel arylomycin analogue with a significantly broadened spectrum activity that enables effective targeting of Gram-negative pathogens.

Figure 4.

Molecular design and development of arylomycin analogue G0775 (14) with the antibacterial profile and results from in vivo efficacy studies against Gram-negative pathogens in mouse models of infection.

In Gram-positive bacteria, SPase is exposed on the surface of the cell where arylomycin antibiotics can readily access this bacterial target. However, in Gram-negative bacteria, the active site of the SPase enzyme is located in the periplasmic space between the cytoplasmic membrane and the outer membrane, and it was initially thought that arylomycins were unable to access SPase from reduced membrane penetration. Interestingly, arylomycins have indeed been shown to penetrate the outer membrane in Gram-negative bacteria, and their lack of activity is the result of a mutation in the Gram-negative SPase LepB target reducing drug-target affinity.⁶⁶

New antibiotic agents capable of effectively treating Gram-negative pathogens are of significant clinical interest as these bacteria often lead to multidrug-resistant infections in humans. To make matters worse, no new class of antibiotic has been approved for the treatment of Gram-negative infections in over 50 years. Smith et al. recently reported the exciting discovery of a novel synthetic arylomycin analogue G0775 (14) that possesses potent antibacterial activities against several Gram-negative ESKAPE pathogens and operates through an unprecedented mechanism of action (Figure 4).⁶⁶

The design of new arylomycin analogues aimed to target Gram-negative pathogens focused on the modification of three structural features, including the (1) N-terminal lipopeptide chain, (2) C-terminal carboxylic acid, and (3) phenolic moieties on the biaryl substructure embedded in the macrocyclic scaffold of 12.⁶⁶ In previous work, crystal structures of arylomycin bound to the Gram-negative SPase LepB indicated that the macrocycle preorganizes the peptide backbone and occupies a conserved region of the substrate-binding pocket; therefore, the macrocyclic architecture was left unaltered during these investigations. Smith et al.⁶⁶ noted that other arylomycin analogues with structural variation of the N-terminal lipopeptide tail modulate activity profiles against Gram-positive bacteria. The N-terminal lipopeptide tail interacts with amino acid residues in LepB that reduces the binding affinity of arylomycin antibiotics; therefore, synthetic modifications to this moiety were of primary interest to engage and optimize binding of the Gram-negative protein.

During these efforts, the most significant improvement toward gaining Gram-negative activity was achieved by replacing the _D-N-Me-Ser-_D-Ala-Gly tripeptide of arylomycin with a diaminobutyric acid unit coupled to a 2-(4-(tert-butyl)phenyl)-4-methylpyrimidine-5 carboxylic acid fragment. In addition, an electrophilic nitrile warhead was installed at the C-terminus carboxylic acid and designed to covalently modify the catalytic serine residue of LepB. The addition of this nitrile warhead improved the activity against several Gram-negative pathogens, including: E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. Final alterations made to the phenolic groups were inspired by previous findings related to arylomycin analogues, demonstrating that such modifications lead to gains in activity. Combined, these changes led to the identification of analogue G0775 (14), which demonstrates ≥500-fold more potent antibacterial activity against Gram-negative pathogens E. coli and K. pneumoniae compared to arylomycin A-C₁₆ (G0775, MIC = 0.125 μg/ mL; arylomycin A-C₁₆, MIC > 64 μg/mL). In addition, G0775 demonstrates excellent antibacterial activities against P. aeruginosa (MIC = 2 μg/mL) and A. baumannii (MIC = 1 μg/mL).⁶⁶

Following initial antibacterial assessment, G0775 (14) was evaluated against 49 multidrug-resistant (MDR) clinical isolates of E. coli and K. pneumoniae. More than half this panel of E. coli and K. pneumoniae clinical isolates was found to be resistant to five, or more, clinically used antibiotics during these investigations. When tested against this panel of MDR isolates, G0775 maintained potent antibacterial activities and reported an MIC ≤ 0.25 μg/mL in 90% of the E. coli and K. pneumoniae isolates. Similar activity profiles were observed when G0775 was evaluated against MDR strains of A. baumannii (MIC ≤ 4 μg/ mL in 90% of 16 MDR strains) and P. aeruginosa (MIC ≤ 16 μg/ mL in 90% of 12 MDR strains). A final challenge was posed in testing G0775 against K. pneumoniae strain CDC 0106, which is known to contain 10 chromosomally encoded and 25 plasmid-encoded genes with resistance to 13 classes of antibiotics. Despite the significant level of resistance elements regarding K. pneumoniae CDC 0106, G0775’s activity remained high when tested against this strain (MIC = 0.5 μg/mL), confirming the activity of this new compound is not affected by an extensive resistance profile.⁶⁶

Target validation confirmed LepB inhibition through whole-cell experiments evaluating G0775 (14) against a bacterial strain that expresses high or low levels of LepB, as controlled by arabinose concentration in growth medium.⁶⁶ In these experiments, G0775 demonstrated increased antibacterial activities with low levels of LepB expression and decreased activities with high expression levels of LepB providing support that G0775 operates through the inhibition of LepB. Spontaneous mutants were generated using G0775 to confirm LepB as the primary target of this new antibacterial agent and gain additional insights. At 8X MIC of G0775 (14), K. pneumoniae and A. baumannii mutants were not detected (mutation frequency below the limit of detection); however, at 16X MIC against E. coli and P. aeruginosa, the frequency of resistance was found to be less than 10^-10. G0775-resistant mutants (spontaneously generated in E. coli) were subjected to targeted and whole-genome sequencing and revealed that resistance-conferring mutations were within LepB’s substrate-binding groove. These results confirmed the whole-cell antibacterial activity of G0775 is operating through the selective inhibition of LepB and that spontaneous resistance to G0775 occurs primarily through mutations to this target.

A cocrystal structure of G0775 bound to LepB provided further support for the spontaneous mutant findings as multiple mutations at critical residues within the substrate-binding groove of LepB closely overlay the binding of G0775 (14). In fact, mutations were found in 8 of the 20 LepB residues that contact G0775. Interestingly, G0775’s nitrile warhead was not found to modify the catalytic serine (S91) as designed; however, the cocrystal structure revealed that this warhead covalently modifies the amine of the catalytic lysine (K146) residue of LepB, resulting in an amidine adduct (Figure 5; see 16). These results were confirmed upon overnight incubation of G0775 (14) with LepB, followed by trypsin digest and LC-MS analysis. Smith et al. noted that the mode of action of the nitrile warhead is novel and could expand the medicinal chemist’s tool kit for future applications in drug design. Enzymology studies of full-length LepB used kinetics to quantify the rate of covalent-bond formation of 14 with the catalytic lysine residue. Results from these kinetic enzyme experiments indicted a tight reversible binding, followed by irreversible inactivation of LepB, confirming a very high affinity of G0775 to this bacterial target.

Figure 5.

Mechanism of action regarding the covalent modification of LepB through the reaction of the nitrile warhead of G0775 (14) reacting with the primary amine of Lys146 (catalytic residue) to form an amidine adduct.

When tested for antibacterial activities in the presence of serum and lung surfactant, G0775 (14) maintained potent in vitro activity, suggesting that this new agent could be used in systemic and pulmonary infections.⁶⁶ With that, G0775 was evaluated for efficacy in mice using multiple infection models against Gram-negative pathogens. Initial experiments investigated G0775 in the murine neutropenic thigh infection (systemic) model in mice and demonstrated potent bactericidal activity (>2-log reduction in colony-forming units) against E. coli ATCC 25922 (1 mg/kg) and K. pneumoniae ATCC 43816 (5 mg/kg). G0775 was also found to demonstrate efficacy against P. aeruginosa and A. baumannii at higher test concentrations (40 m g/kg) in the neutropenic thigh model, which aligned with potency profiles from initial MIC experiments against these pathogens.

Using a lung infection model in mice, G0775 (14) demonstrated a dose-dependent in vivo efficacy against the MDR strain CDC 0106 (2 mg/kg G0775, observed bacteriostatic activity; 20 mg/kg G0775, bactericidal activity was observed in this lung infection model). In a final assessment, G0775 was examined for protection of mice from a lethal challenge of K. pneumoniae Z strain ATCC 43816 in a mucin peritonitis model. G0775 delivered a dose-dependent increase in survival with two treatments of 5 mg/kg on day zero and was able to completely protect mice from death for 84 h (all vehicle- only treated mice died by the 60 h time point in this experiment). These in vivo efficacy findings are very promising and suggest that G0775 (14) could be useful in the treatment of multiple Gram-negative pathogens, including MDR infections.⁶⁶

TARGETING MUTANT DNA GYRASE AND RESCUING MICE FROM FLUOROQUINOLONE-RESISTANT MRSA WITH DEOXYNYBOMYCIN ANALOGUES

Deoxynybomycin (DNM, 24) is an interesting natural product antibiotic that demonstrates potent activities.^68,69 Following the initial studies, DNM was later reported to display significantly enhanced activities against fluoroquinolone-resistant, methicillin-resistant Staphylococcus aureus (MRSA) bearing the S84L mutation in GyrA of DNA gyrase.⁷⁰ Unfortunately, the isolation of DNM from natural sources has been troublesome, and the initial total synthesis of this antibiotic was very low yielding. DNM also suffers from very low solubility (only soluble in concentrated acid), limiting biological studies and preclinical studies.

Excited by the potential to deliver a promising new agent to target fluoroquinolone-resistant MRSA, Hergenrother and co-workers developed a modular total synthesis of DNM and related analogues aimed to address solubility limitations regarding the parent natural product (Figure 6A).⁶⁹ A series of 15 analogues were designed and synthesized to systematically probe four positions on the DNM scaffold (this synthetic route parallels chemistry utilized to access deoxynyboquinone, a related natural product with anticancer activities^71,72). The resulting DNM analogues were then subjected to antibacterial testing against wild-type S. aureus (ATCC 29213, WT) and fluoroquinolone-resistant S. aureus (NRS3, FQR). DNM served as the benchmark for assessing new synthetic analogues and demonstrating good antibacterial activities against wild-type S. aureus (MIC = 2 μg/mL, ATCC 29213) with an impressive 67-fold increase in antibacterial potency against fluoroquinolone-resistant S. aureus (MIC = 0.03 μg/mL, NRS3; Figure 6B).⁶⁹

Figure 6.

(A) Chemical synthesis of DNM (24) and related analogues for antibacterial assessment. (B) Structure–activity relationship analysis along with interesting DNM agents.

Strategically, the synthetic analogues of DNM (24) were designed to prevent optimal π-stacking interactions, which was thought to be the primary factor influencing poor water solubility. Modifications to each of the four positions of the DNM scaffold provided a clear structure—activity relationship (SAR) profile that favored small structural alterations to maintain antibacterial activities (Figure 6B).⁶⁹ Analogues DNM-2 (25) and DNM-5 (not shown) had single methyl group additions at two different positions on the nitrogen-bearing side of the DNM scaffold and maintained the same activity profiles and potency against fluoroquinolone-resistant S. aureus compared to the parent DNM; therefore, small changes were well-tolerated. However, the remainder of the synthetic analogues of DNM contained methyl groups at the other positions or had modifications that increased steric bulk (mainly straight-chain alkyl groups) that negatively impacted anti-bacterial activities.

Two of the most potent DNM analogues (DNM-2, 25; DNM-8, 26) were evaluated against a panel of MRSA and vancomycin-resistant Enterococcus (VRE) clinical isolates.⁶⁹ All MRSA and VRE strains that were sensitive to DNM, DNM-2, and DNM-8 were resistant to ciprofloxacin (27; fluoroquinolone antibacterial agent). These MRSA strains were confirmed to have the same S84L mutation in GyrA (by sequencing), which drives the enhanced antibacterial activities of DNM and analogues. The VRE isolates contained two different substitutions for GyrA (S83I or S83R), which impacted their sensitivity toward DNM (S83I mutation very sensitive, MIC = 0.125–1 μMg/mL; S83Rless sensitive, MIC ≥ 1 μg/mL). During these investigations, the activity profiles for DNM (24), DNM-2 (25), and DNM-8 (26) closely aligned against the panel of MRSA and VRE isolates.

Following the assessment against clinical isolates, DNM (24) and DNM-2 (25) demonstrated inhibition of mutant DNA gyrase using a rigorous series of dose-dependent and time-dependent in vitro DNA cleavage assays comparing inhibitory activity profiles with ciprofloxacin against both wild-type and mutant DNA gyrase.⁶⁹ The in vitro experiments against mutant DNA gyrase were found to be consistent with the activity profiles obtained for DNM and analogues against the panel of MRSA and VRE clinical isolates, supporting the importance of a mutant DNA gyrase to sensitize these pathogens to DNM. Resistance studies in S. aureus (ATCC 29213) demonstrated that the development of high-level ciprofloxacin-resistant strains (CIP MIC = 16–64 μg/mL), similar to what is seen in the clinic, takes multiple steps to develop and achieve the key S84L mutation in GyrA. These high-level CIP-resistant mutants were extremely sensitive to DNM (MIC = 0.03–0.06 μg/mL) and DNM-2 (MIC = 0.06–0.12 μg/mL). Additional studies showed that DNM resistance in CIP-resistant strains was rare (mutation frequencies at ≥10⁻¹⁰) and that DNM-resistant S. aureus strains demonstrated significantly improved sensitivity toward ciprofloxacin (MIC = 0.25–8 μg/mL). All DNM-resistant strains had reverted to WT GyrA (Ser84), completing a cycle of complementary resistance/sensitivity of ciprofloxacin and DNM (Figure 7), an incredibly unique activity profile of clinical importance for fluoroquinolone-resistant MRSA and VRE.

Figure 7.

Resistance/susceptibility cycle from ciprofloxacin (27) resistance and DNM (24) susceptibility to DNM resistance and ciprofloxacin susceptibility.

When advanced to animal studies in mice, DNM-2 (25) was found to be well-tolerated and no signs of toxicity were observed at the highest dose administered (50 mg/kg; oral gavage).⁶⁹ Pharmacokinetic (PK) studies demonstrated that DNM-2 showed excellent bioavailability with peak serum concentrations of 42.6 μM (12.8 μg/mL) and an area under the curve of 44 h μg/mL, following an oral dose of 50 mg/kg. These bioavailability parameters were also assessed with DNM (24) and DNM-3 (structure not shown), and the results from these compounds aligned with their diminished aqueous solubility profiles compared to DNM-2 (25).

Following toxicity and pharmacokinetic assessment, DNM-2 (25) was advanced to in vivo efficacy studies in a mouse infection model. During these studies, mice were infected with fluoroquinolone-resistant MRSA (NRS3; tail vein injection) and treated with DNM-2 (50 mg/kg; oral gavage), ciprofloxacin (50 mg/kg; oral gavage), or vehicle control once a day for 10 days. A Kaplan-Meier survival curve showed a significant difference in survival rates between DNM-2 (25) treated mice (>90% survival) and both ciprofloxacin (>30% survival) and vehicle (<30% survival) treatments.⁶⁹

In continued efforts from the Hergenrother lab, DNM analogue 6DNM-NH3 (not shown) was developed using an innovative strategy aimed to convert Gram-positive only antibacterial agents into compounds with broad-spectrum activities.^73,74 Gram-negative bacteria have two cellular membranes (the outer membrane is coated with lipopolysac-charides) that pose a significant challenge for small molecules to transverse, and many compounds that are able to enter are rapidly removed via efflux pumps before binding their corresponding bacterial target. Hergenrother and co-workers investigated compound accumulation in E. coli using an extensive series of diverse compounds and were able to establish new guidelines for small molecules to enter Gram-negative bacterial cells (via porins) more rapidly than they are removed from bacterial cells via efflux pumps. These guidelines are referred to as the “eNTRy rules” and combine structural features that include: (1) having a primary, or nonsterically hindered, amine group, (2) containing nonpolar groups (common in organic molecules), (3) bearing a rigid structure with ≤5 rotatable bonds, and (4) having a low globularity score.⁷³ With these guidelines, 6DNM-NH3 was designed, chemically synthesized, and demonstrated high levels of compound accumulation in E. coli and broad-spectrum antibacterial activities against several Gram-negative bacterial pathogens, as predicted by these newly established “eNTRy rules”.

OVERCOMING RESISTANCE TO THE “ANTIBIOTIC OF LAST RESORT” WITH INNOVATIONS IN THE DESIGN OF VANCOMYCIN ANALOGUES

Vancomycin (10) is a glycopeptide antibiotic that has been in clinical use for over 60 years and is referred to as the “antibiotic of last resort” for its effectiveness in treating methicillin-resistant S. aureus (MRSA) infections and other resistant Gram-positive pathogens.⁷⁵ The incredibly complex molecular architecture of vancomycin enables selective binding to C-terminus _D-Ala-_D-Ala residues of peptidoglycan precursors through a network of hydrogen bonding interactions that results in the inhibition of cell wall synthesis and bacterial death.⁷⁶ Vancomycin resistance was initially observed in Enterococci (VRE isolates) several decades after being introduced in the clinic; however, vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) infections have recently emerged.⁷⁷

Bacteria gain resistance to vancomycin through late-stage remodeling of peptidoglycan precursors that leads to the replacement of the terminal _D-Ala-_D-Ala residues with _D-Ala-_D-Lac.^78,79 This remodeling event in vancomycin-resistant bacteria results in the loss of a critical N—H hydrogen bond donor of the terminal D-alanine residue and replacement with a repulsive oxygen atom from _D-lactate. The altered _D-Ala-_D-Lac residue demonstrates a ~1000-fold decrease in binding affinity between

vancomycin and target residues, corresponding to observed losses in antibiotic activity (via assessment in MIC assays). Due to the emergence of vancomycin-resistant bacteria (VRE, VRSA), there is an urgency to develop new antibiotics that are able to overcome this resistance mechanism. Despite the few semisynthetic glycopeptide antibiotics used in the clinic (e.g., televancin), none of these agents address the late-stage remodeling of peptidoglycan precursors as the underlying molecular basis for vancomycin resistance.

Total syntheses of vancomycin, or vancomycin aglycon, were reported by the Evans,⁸⁰ Nicolaou,⁸¹ and Boger⁸² laboratories in the late 1990s.⁷⁷ Significant advances from the Boger lab have enabled the semisynthesis of vancomycin analogues designed to address the late-stage peptidoglycan remodeling from _D-Ala-_D-Ala to _D-Ala-_D-Lac.^78,79,83,84 In 2011, Boger and co-workers reported a vancomycin analogue engineered for dual binding to _D-Ala-_D-Ala and _D-Ala-_D-Lac residues through the installation of a deep-seated amidine group, countering the single atom exchange (NH to O) to reinstate molecular recognition, target binding, and potent antibacterial activity⁷⁸ (note: the initial amidine was reported as the aglycon analogue; the structure is not shown here). More structurally advanced analogues were later reported bearing the dual-binding amidine group and sugar moieties of vancomycin (note: sugar moieties were installed from the aglycon using sequential enzymatic glycosylation reactions; see compound 28, Figure 8).⁸³

Figure 8.

Vancomycin’s structure and drug-target interactions that lead to potent antibiotic activities or resistance. In addition, new vancomycin analogues 28–30 that have been designed to overcome resistance or induce new mechanisms of action by Boger and co-workers ⁸⁵are presented here.

In the same report, an additional vancomycin analogue was synthesized bearing the amidine group for dual binding and a (4-chlorobiphenyl)methyl group was installed on the aminosugar moiety via reductive amination to yield 29.⁸³ The 4- chlorobiphenyl (CBP) substitution of the peripheral carbohydrate has been shown to promote antibiotic dimerization and membrane anchoring in semisynthetic glycopeptide antibiotics, including televancin. The CBP group is believed to induce an independent mechanism of action as glycopeptides containing this moiety with impaired _D-Ala-_D-Ala binding properties still exhibit antibacterial activities. As designed, each of the amidine-based vancomycin analogues demonstrated impressive antibacterial activities to vancomycin-resistant strains with the CBP- bearing analogue showing significantly enhanced potencies (28, MIC = 0.5 μg/mL against VRE strains; 29, MIC = 0.005 μg/mL against VRE strains; Figure 8).

In 2017, Boger and co-workers reported a new series of vancomycin analogues that combined modifications to provide a dual _D-Ala-_D-Ala/_D-Ala-Lac peptidoglycan residue binding with C-terminal peripheral amide modifications containing a quaternary ammonium salt.⁸⁵ A methylene pocket-modified vancomycin analogue (the most synthetically accessible material during these studies; exhibits modest dual _D-Ala-_D-Ala/_D-Ala- Lac binding affinity and antibacterial activities against vancomycin-resistant strains) was subjected to amide coupling to rapidly install the quaternary ammonium side chain, which was shown to induce cell wall permeability independent of the inhibition of cell wall biosynthesis. This modification delivered vancomycin analogues with ~200-fold improvements in antibacterial potency.

During these studies, a series of analogues was designed and synthesized to investigate the impacts that various structural modifications had on antibacterial activity. Vancomycin analogue 30 was synthesized and combined the (1) methylene pocket modification for dual binding of _D-Ala-_D-Ala/_D-Ala-Lac peptidoglycan residues, (2) peripheral amide modifications bearing a quaternary ammonium salt, and (3) CBP moiety resulting in outstanding potency against VRE strains (MIC = 0.005—0.01 μg/mL; Figure 8).⁸⁵ In addition, 30 reported no resistance development against the VRE strain VanA E. faecium (ATCC BAA 2317) after 25 daily passages at0.5X MIC and only a 4-fold increase in the MIC value after extending this assessment to 50 daily passages. Related analogues bearing only one or two of these synthetic modifications were tested alongside 30, and resistance development was significantly increased for these analogues depending on the number of key structural modifications that induced different modes of action (related vancomycin analogues with one structural modification resulted in a 64—128-fold increase in MIC values after 50 daily passages; two structural modifications resulted in a 8—16 -fold increase in MIC values after 50 daily passages). Vancomycin analogues bearing the three key modifications (one could imagine an amidine analogue being incorporated in combination with the peripheral modifications of 30) are expected to display durable antibacterial activity that would avoid rapidly acquired antibiotic resistance in the clinic.

The Boger lab most recently reported a series of N-terminus alkylated analogues accessed through alkylation (reaction with methyl iodide) or reductive amination reactions (e.g., 31–33; Figure 9).⁸⁶ During these investigations, reductive amination was performed in a site-selective fashion at the N-terminus amine of vancomycin with different aldehydes and sodium cyanoborohydride in the presence of a weak acid to avoid a competing reductive amination reaction of the vancosamine sugar moiety (installation of the CBP group occurs via the reductive amination at the primary amine of the vancosamine under basic conditions). The N-terminus modifications to vancomycin, and analogues bearing additional peripheral modifications, were found to be well-tolerated in antibacterial assays providing further insights into the SAR related to vancomycin analogues; however, these modifications were not found to increase antibacterial activities.

Figure 9.

Recent vancomycin analogues 31−33 bearing an N-terminus modification and an overview of the significant advances made regarding innovations in vancomycin analogue design to overcome resistant bacteria and create durable antibiotic compounds.

Over the past decade, the Boger lab has made significant progress related to the design and development of exciting and promising new vancomycin analogues. ^{75,77−79,82−87} These impressive innovations have enabled the engineering of new vancomycin analogues that effectively bind both _D-Ala-_D-Ala (sensitive) and _D-Ala-_D-Lac (resistant) peptidoglycan residues and incorporate peripheral modifications at multiple positions of this antibiotic’s complex structure to dramatically increase antibacterial potency through independent mechanisms of action (membrane anchoring, membrane permeability). We are encouraged by the several advances related to new vancomycin analogues that Boger and co-workers have engineered through molecular design and synthetic skill. Finally, we look forward to these promising designs and/or agents making a significant impact to the treatment of vancomycin-resistant infections in the clinic.

REVITALIZING THE OPTOCHIN SCAFFOLD TO IDENTIFY NARROW-SPECTRUM INHIBITORS OF ATP SYNTHASE IN S. pneumoniae

Optochin (35) is a semisynthetic derivative of the cinchona alkaloid quinine (34) that possesses narrow-spectrum activity against Streptococcus pneumoniae and has been used to treat septicemia in humans.⁸⁸ S. pneumoniae is a Gram-positive pathogen that is the leading source of bacterial pneumonia and meningitis around the world. The pneumococcal vaccine PCV13 provides immunity to 13 of the most prevalent S. pneumoniae serotypes and has significantly reduced pneumococcal disease worldwide. However, widespread use of PCV13 and the vaccine PCV7 has led to increased incidences of drug-resistant S. pneumoniae infections, complicating therapy for those unvaccinated or infected with one of the 80 serotypes not covered by these vaccines.⁸⁹

Despite optochin’s narrow-spectrum activity against S. pneumoniae, this antibiotic was eventually discontinued as a result of toxicity and its moderate efficacy in treating lobar pneumonia, the most prevalent infection caused by this pathogen.⁸⁹ During clinical use in humans over one hundred years ago, optochin (35) was found to be effective early in the course of treating pneumonia infections; however, S. pneumoniae appeared to become resistant during treatment. The lack of a sustained therapeutic effect of optochin is now attributed to suboptimal drug exposure due to a lack of pharmacokinetic (PK) and pharmacodynamic (PD) information, which could have informed a dosing strategy to achieve optimal drug exposures.

Unfortunately, optochin (35) has largely remained forgotten over the last century. However, with the emergence of resistant S. pneumoniae isolates coupled to the need for effective new therapeutic options against this pathogen, Aldrich and co-workers recently reported the chemical synthesis, microbiological investigation, and initial PK assessment of a new series of optochin derivatives (Figure 10).⁸⁹ Previous genetic studies show optochin-resistance maps to atpE, which encodes ATP synthase’s C-ring in S. pneumoniae. ATP synthase plays a critical role in energy metabolism, synthesizing the majority of ATP in bacteria, and is now a validated antibacterial target. Recently, the FDA approved bedaquiline⁹⁰ (a quinoline drug, structure not shown) that inhibits ATP production through the selective binding of the C-ring of ATP synthase in Mycobacterium tuberculosis (Mtb). Aldrich and co-workers also noted that optochin (clog P 3.1, MW 340) has considerably more favorable physicochemical properties compared to bedaquiline (clog P 7.3, MW 556), making this semisynthetic antibiotic an ideal scaffold for the development of new ATP synthase inhibitors.⁸⁹

Figure 10.

Optochin (35), a semisynthetic derivative of quinine, has previously been used for the treatment of S. pneumoniae infections. Recently, a report by Aldrich and co-workers ⁸⁹ describes the synthesis, SAR, and biological investigations of a series of new optochin derivatives that target ATP synthase. Resistance profile notes. DS: drug-sensitive (strain); A: amoxicillin; P: penicillin; C: cefotaxime; E: erythromycin; T: tetracycline; Ch: chloramphenicol; M: meropenem; Er: ertapenem; Mo: moxifloxacin.

Aldrich and co-worker’s initial efforts were aimed to systematically define the impact various structural features have on optochin’s antibacterial activity through chemical synthesis and biological investigations of more than 50 related analogues.⁸⁹ Each newly synthesized optochin analogue was evaluated for antibacterial activity in MIC assays against S. pneumoniae strain D39 and tested against HepG2 cells to determine mammalian cytotoxicity. The structure-activity relationship studies began by probing the C-6’ position of optochin (see 36; Figure 10). In 1911, it was reported that the ethyl group was the optimal substituent at C-6’ of optochin and indicated that an extensive series of homologues were evaluated and found to be less active; however, upon further analysis of previous findings, a significant amount of this work and related characterization data were missing. Several modifications at C-6’ of the optochin scaffold were carried out via the following three- step synthetic pathway, starting from commercially available quinine: (1) hydrogenation to reduce the olefin at C-3 (H₂, Pd-C; 97% yield), (2) demethylation at C-6’ to the corresponding phenol using sodium thioethoxide (EtSH, NaH; 79% yield), and (3) final alkylation of the phenolic hydroxyl group at C-6’ with a primary or secondary alkyl bromide or iodide in the presence of cesium carbonate (R-X, Cs₂CO₃; X = Br, I; 9–87% yield).

Additional synthetic modifications were made at the C-3 position of optochin (35) utilizing a ruthenium-catalyzed isomerization of the olefin moiety, giving a 3:1 E/Z isomer ratio of the products (83% yield). Interestingly, the olefin isomerization alone dramatically increased antibacterial activity 32-fold during these investigations when tested alongside quinine (34).⁸⁹ The resulting olefin was utilized to afford several other analogues through oxidative cleavage (resulting ketone not shown) and synthesized downstream analogues; however, the isomerized olefin intermediate gave rise to the new lead compound from this series following the previously mentioned (1) demethylation of C-6’ (EtSH, NaH, DMF; 87% yield) and (2) alkylation of C-6’ with bromocyclobutane (with Cs₂CO₃, DMF; 79% yield).

A final series of optochin derivatives were synthesized to probe the stereochemical requirements at C-8 and C-9 along with defining the importance of the alcohol group.⁸⁹ It was found that inversion of either stereocenter was not tolerated and these changes completely obliterated antibacterial activities, validating the strict structural requirements for the spatial orientation of these groups at C-8 and C-9. In addition, bioisosteric replacement of the 9-hydroxyl group with a fluorine atom (with inverted stereochemistry in 9S-configuration) also resulted in a complete loss in activity, demonstrating the importance of the C-9 alcohol to optochin’s antibacterial activity.

The SAR knowledge gained during the initial investigations informed synthetic efforts of more advanced optochin analogues later in these studies. Aldrich and co-worker’s efforts led to the discovery of compound 37, which demonstrates 4- to 16-fold more potent antibacterial activities against drug-sensitive S. pneumoniae strains (MIC = 0.25–0.5 μg/mL) and multidrug-resistant isolates (MIC = 0.25–1 μg/mL) when compared to optochin 35.⁸⁹ In addition, compound 37 demonstrated a narrow-spectrum activity profile against S. pneumoniae, similar to optochin, by reporting significantly elevated MIC values (≥64- fold) against other bacterial pathogens. Compound E-37 reported a selectivity index of 93 (HepG2 cytotoxicity, CC₅₀ = 65 μM; selectivity index = CC₅₀/MIC; E-37, MIC = 0.25 μg/mL = 0.7 μM), corresponding to very good targeting of S. pneumoniae (note: compound 37 was tested as a mixture of E/Z isomers in some experiments but also as pure E and Z isomers).

Initial target validation experiments utilized a polymerase chain reaction (PCR)-based approach to generate point mutations of atpE in S. pneumoniae strain R6 (see R6-atpE_G142C and R6-atpE_G145A), which allowed Aldrich and co-workers to generate two optochin-resistant strains for assessment.⁸⁹ When tested against these S. pneumoniae mutant strains, the antibacterial activities of optochin (35) and compound 37 were significantly decreased, as MIC values for these agents shifted 16- to 32-fold confirming AtpE as the putative molecular target (Figure 10). Further target validation studies were performed by sequencing atpE of 29 mutants generated from 4, 8, and 16X MIC treatments with compound 37. Each of the 29 mutants contained missense mutations in the atpE gene, as predicted, and the five mutants sequenced from the 16X MIC treatments of 37 mapped to F50L and F45C within the C- terminal helix of AtpE (Figure 11). As a comparator, 15 mutants resistant to optochin 35 were sequenced and mutations were mapped to similar positions of AtpE.

Figure 11.

ATP synthase is a multimeric complex that plays a central role in energy metabolism and intracellular pH homeostasis. Compound 37 targets the C-ring for inhibition of the ATP synthase in S. pneumoniae.

ATP synthase is believed to be involved in pH homeostasis in S.pneumoniae, similar to other fermentive microorganisms, by moving acid out of the cell in an ATP-dependent manner. Aldrich and co-workers investigated the impact compound 37 has on S. pneumoniae and found this ATP synthase inhibitor to rapidly decrease the pH in a time- and concentration-dependent fashion using a pH-sensitive ratiometric GFP biosensor on a plasmid, allowing noninvasive pH measurements.⁸⁹ Therefore, it was concluded that the mode of action of 37 is the disruption of pH homeostasis, likely through ATP synthase inhibition in S. pneumoniae. These exciting findings have revitalized optochin’s scaffold for the development of narrow-spectrum agents against S. pneumoniae and may offer significant therapeutic benefits to patients with pneumococcal infections.

DEVELOPMENT OF FULLY SYNTHETIC GROUP A STREPTOGRAMIN ANTIBIOTICS

Streptogramin antibiotics⁹¹ are produced by several Streptomyces species and comprise two distinct chemotypes: group A streptogramins are 23-membered macrocyclic polyketide/non- ribosomal peptide hybrids, and group B streptogramins are 19- membered macrocyclic depsipeptides.⁹² Group A and B streptogramins function synergistically and in concert with one another to target adjacent binding sites in the bacterial ribosome’s catalytic center, which inhibits protein synthesis.⁹³ Streptogramin antibiotics demonstrate potent antibacterial activities against Gram-positive bacteria, including drug-resistant strains; however, these agents have limited clinical use as a result of their resistance liabilities and problems with water solubility.⁹²

Fermentation of the streptogramin antibiotics virginiamycin M1 (11), virginiamycin M2 (41), and pristinamycin IA (40) has led to successful semisynthesis campaigns, yielding clinical agents with improved water solubility (e.g., dalfopristin 38, synthesized from virginiamycin M1; quinupristin 39, synthesized from pristinamycin IA; see Figure 12).⁹² In 1999, the FDA- approved Synercid as an antibiotic therapy, combining the semisynthetic streptogramins dalfopristin and quinupristin to treat patients with multidrug-resistant skin and skin-structure infections or vancomycin-resistant Enterococcus faecium bacteremia. Synercid has limited clinical use as an IV-only formulation and a relatively narrow spectrum of activity. In addition, an orally bioavailable combination of flopristin-linopristin (42 and 43, semisynthetic streptogramins) named NXL-103 underwent phase-II clinical trials in 2011 but has not progressed further.⁹²

Figure 12.

Chemical structures for select streptogramin antibiotics. Group A and B streptogramins perform their respective modes of action in concert with one another to inhibit bacterial ribosomes. Semisynthetic efforts have previously led to one clinically used antibiotic treatment (Synercid; combination of dalfopristin 38 and quinupristin 39) and another clinical candidate (NXL-103; combination of flopristin 42 and linopristin 43).

Despite progress advancing streptogramin analogues toward clinical applications, semisynthetic approaches do not allow broad exploration of structure—activity relationships of these complex antibiotics. Li and Seiple have recently reported total synthesis approaches that enable structural modifications to be made at multiple positions of Group A streptogramin antibiotics (Figure 13).^92,93 A goal of this work is to provide gram quantities of multiple, fully synthetic Group A streptogramin antibiotics and design new analogues featuring optimized drug-target interactions guided by crystallographic data of streptogramin analogues bound to bacterial ribosomes.

Figure 13.

Convergent and modular synthetic routes reported by Li and Seiple ^92,93 enable rapid access to several fully synthetic Group A streptogramin antibiotics.

In 2017, Li and Seiple reported the total synthesis of madumycin I (55), madumycin II (56), virginamycin M2 (11), and virginamycin Ml (41) using a convergent and modular approach to access these Group A streptogramin antibiotics.⁹² Key asymmetric reactions were performed on large scale early in the synthetic sequence to establish multiple stereocenters, including: (l) the Mukaiyama-type vinylogous Aldol reaction using a proline-derived boron catalyst (see synthesis of 47; 94% yield, 87% ee; >1 g scale; Figure 13) and (2) the Aldol reaction using acetal thiazolidinethione 49 in the presence of titanium- (IV) chloride (see synthesis of 51; 64% yield, single diastereomer; multigram scale). The thiazolidinethione auxiliary of 51 was later displaced by the organolithium species generated from oxazole 52. The assembly of the macrocyclic scaffold of the targeted Group A streptogramins occurred through a HATU-mediated amide coupling of key “left half’ 48 and “right half’ 53 fragments (88% yield), followed by the crucial Stille macro-cyclization step from 54 en route to streptogramin antibiotics madumycin I (55) and madumycin II (56).

During these initial total synthesis efforts, the key intra-molecular Stille step required considerable optimization, and it was found that 20 mol % Pd₂(dba)₃ with the sterically hindered JackiePhos (phosphine) ligand provided a 64% yield of the target compound.⁹² JackiePhos was designed by Buchwald and co-workers to facilitate challenging transmetalations and proved useful for this route.⁹⁴ Following macrocyclization of 54, silyl group removal was afforded with the use of tetrabutylammonium fluoride (TBAF) to yield madumycin I (55, 94% yield; >1.5 g of this antibiotic). Madumycin I was also reduced with sodium borohydride in the presence of diethylmethoxyborane to yield madumycin II (56, 72% yield) as a single diastereomer. The modularity of this synthetic route was demonstrated by synthesizing virginiamycinM1 (11) and virginiamycin M2 (12), utilizing the same strategy with slight modifications to accommodate structural diversity of the amino acid moiety embedded in the macrocyclic scaffold of these streptogramins.

In 2019, Li and Seiple reported a second-generation synthesis to virginiamycin M2 (41) aimed to address limitations experienced in their initial work, such as (1) acid-sensitive synthetic intermediates, (2) reliance on organotin reagents (known toxicity issues), and (3) a moderate yielding Stille macrocyclization reaction.⁹³ To overcome these synthetic limitations, a revised route was developed to capitalize on the initial steps from the first-generation route yet incorporate a ring-closing metathesis (RCM) reaction for the critical macro-cyclization step. By targeting intermediate 61 for RCM, undesirable organostannane intermediates were not required and the focus turned to implementing a successful and high-yielding RCM reaction.

Initial RCM attempts to close the 23-membered ring of virginiamycin’s architecture using Grubbs I/II and Hoveyda-Grubbs I/II catalysts at room temperature resulted in no conversion (by NMR analysis), while elevated temperatures continued to give no yield of target compounds. With persistence and effort, the first promising RCM result came with the phosphine-free Hoveyda-Grubbs II catalyst, resulting in a 15% yield of the desired macrocycle (structure related to virginiamycin M2, not shown).⁹³ Additional work to investigate a panel of ruthenium catalysts (Grubbs III, Piers II, Zhan 1B, Grela II 62, three other Ru catalysts) utilized in the metathesis reaction with a structurally related model substrate of interest led to several examples of improved RCM reactions with yields of 25–49%. However, the best substrate for this RCM reaction led directly to virginiamycin M2 41 in 72% yield upon ring-closure using 2 × 8 mmol % (batchwise addition) Grela II (ruthenium) catalyst 62 in dichloromethane at room temperture (Figure 13).

Collectively, this modular chemistry enables rapid access to sufficient quantities of fully synthetic Group A streptogramin antibiotics. These advances will enable a pathway for significantly more chemical diversity to be incorporated to these antibiotic scaffolds compared to previous semisynthetic efforts. In addition, this total synthesis platform can be used to rationally design new analogues to overcome resistance through the optimization of streptogramin agents.

CAROLACTON: A NARROW-SPECTRUM ANTIBIOTIC THAT TARGETS S. mutans BIOFILMS

Carolacton (63) is an interesting, narrow-spectrum antibiotic identified from the myxobacterium Sorangium cellulosum for its potent antibacterial activities against E. coli strain tolC (MIC = 0.06 μMg/mL).⁹⁵ During the initial investigations, carolacton was found to be inactive against other planktonic bacteria (MIC > 40 μMg/mL); however, it proved to significantly reduce viable Streptococcus mutans biofilm cells, killing 35% of biofilm cells at 0.005 μMg/mL and 66% of biofilm cells at 0.025 μg/mL. These findings are of significance to human health as S. mutans plays a major role in caries-associated (tooth decay)⁹⁶ and endocarditis- associated infections (Figure 14).⁹⁵

Figure 14.

Chemical structures of carolacton 63 and related synthetic analogues 64–69. 99–104

Subsequent investigations demonstrated that carolacton (63) promotes cellular membrane defects in S. mutans biofilms in lower pH environments;^95,97,98 however, additional work regarding carolacton’s mechanism of action did not adequately address why this natural product targets S. mutans biofilms. Carolacton has a complex polyketide-type structure composed of eight stereogenic centers and 12-membered macrolactone. With the significant biological activity profile, carolacton has received considerable interest from the synthetic chemistry community, and impressive total syntheses have been reported by the Kirschning,⁹⁹ Wuest and Phillips,¹⁰⁰ and Goswami¹⁰¹ groups. Each of these total synthesis efforts have required 19 to 27 total steps to yield carolacton.

An initial series of carolacton analogues was reported by Kirschning and co-workers to gain structure—activity relationship (SAR) insights (see analogues 64—66, Figure 14).¹⁰² Alterations in ring size, oxidation at C-17 (to ketone 66), and inversion of the stereocenter at C-9 of carolacton led to abolished activity, demonstrating the intricate structural requirements related to the macrolactone moiety for this antibiotic to elicit its activity. Despite these new analogues being inactive, findings from these initial studies pointed to the specificity requirements likely for drug—target interactions between carolacton and an unknown biological target.

Additional work to investigate side-chain variants was pursued by Wuest and co-workers using a diverted total synthesis (DTS) approach to access simplified carolacton analogues for biological investigations.^103,104 For these studies, carolacton analogueswere designed to incorporate the macrolactone unit primarily unaltered to avoid losses in activity, on the basis of the initial SAR findings by Kirschning and co-workers.¹⁰² Initial DTS efforts to by the Wuest lab yielded 16 new carolacton analogues for microbiological assessment.¹⁰³ From these efforts, carolacton analogues were identified to display differential biofilm phenotypes, which include: (1) inhibition of S. mutans biofilm formation, (2) inhibition of biofilm maturation, and (3) inducing acid-mediated cell death during biofilm formation, similar to carolacton 63. These investigations led to the identification of C3 (67), an analogue bearing a simplified aryl isostere side chain (replacing the trisubstituted alkene of carolacton) that required only 10 steps to synthesize. C3 demonstrated selective S. mutans biofilm inhibition (minimum biofilm inhibitory concentration, MBIC₅₀ = 63 μM; MIC > 500 μM). In addition, analogue D4 (68, also referred to as “carylacton”) was found to have activity similar to carolacton 63 by inducing the acid-mediated death of S. mutans biofilm cells in confocal microscopy experiments (Figure 14).¹⁰³

In a second-generation study of carolacton analogues bearing a simplified side chain, the Wuest lab identified compound (+)-2 (69, Figure 14) and reported an MIC = 250 μM against planktonic S. mutans cells and an IC₅₀ = 44 μM against S. mutans biofilm formation.¹⁰⁴ In addition, LIVE/DEAD staining showed that treating S. mutans biofilms with (+)-2 led to 76% biofilm cell death (at 125 μM) and 59% biofilm cell death (at 63 μM). Treatment with analogue (+)-2 resulted in a 75% reduction (at 62 nM) and 93% reduction (at 2 μM) of viable S. mutans biofilm cells (determined by colony forming unit counts), which proved to be more potent than carolacton (30% CFU/mL reduction at 62 nM and 78% CFU/mL reduction at 2 μM when tested alongside (+)-2).

Wuest and co-workers noted that carolacton s activity is dependent on a drastic drop in pH that is observed during biofilm formation. Following initial biological assessment, analogue (+)-2 (69) was advanced to mechanistic studies to answer questions as to why carolacton specifically targets S. mutans biofilms. Initial mechanistic experiments measured the susceptibility of preacidified planktonic cultures to (+)-2 and found this carolacton analogue to inhibit planktonic cells with an IC₅₀ ~ 10 μM.¹⁰⁴

The planktonic inhibitory activity of (+)-2 (69) was then used in a forward chemical genetic approach to determine how this compound targets the acid tolerance response (ATR) in S. mutans (Figure 15). After the assessment of an extensive library of S. mutans mutants (with known phenotypic profiles), Wuest and co-workers selected 17 mutants that were hypothesized to be associated with ATR. Collectively, this group contained genes responsible for S. mutans (1) acid tolerance mechanisms, (2) two component systems, (3) cell division, (4) cellular regulation, and (5) glucan synthesis. Each mutant strain was treated with 125 μM of (+)-2 (69), and viability was measured. Results from these experiments showed that 14 of the 17 mutant strains were susceptible to (+)-2 (69) compared to the WT S. mutans strain (UA159); however, two mutants, SMU_484 (ApknB) and SMU_1276c (AezrA), were found to be more susceptible.¹⁰⁴ The activity of (+)-2 (69) was significantly reduced against the SMU_1591 mutant compared to the WT S. mutans strain, demonstrating this transcriptional regulator is partially responsible for the inhibition patterns observed for this compound. SMU_1591 is deficient in the gene that codes for carbon catabolite protein A (CcpA) and is a transcriptional regulator that controls carbon usage within the bacterial cell and regulates several downstream pathways associated with ATR in S. mutans, including: EPS formation (ftf, gtBC), cell attachment (fruA), acetate metabolism (pta, ack), branched-chain amino acid synthesis (ilvCE), oxidative stress tolerance (cid, lrg), and other virulence mechanisms (Figure 15).

Figure 15.

Mechanistic studies of carolacton analogue (+)-2 (69) in a chemical genetic screen reveal CcpA as the putative target in S. mutans. Background items noted for the bottom portion of the figure: upper left, bacterial cells surrounded by hydrogen peroxide molecules; lower left, acetyl- CoA molecule; upper right, structures of valine and leucine; lower right, EPS formation and surface-attached biofilm.

With the considerable amount of work related to carolacton 63, analogue (+)-2 (69) has been an important compound for several reasons, including: (1) an increased potency against S. mutans biofilms, (2) a significantly reduced synthetic route to access this analogue, and (3) the planktonic activity of analogue (+)-2 (69) enabling a screen against a mutant library to identify CcpA as a putative target.¹⁰⁴ Additional work in this area will aim to answer additional questions related to carolacton’s biological activity, multispecies biofilm communities, and oral pathogens. Carolacton is a very exciting narrow-spectrum antibiotic and could lead to significant breakthroughs toward the treatment of S. mutans biofilms.

PHENAZINE ANTIBIOTIC INSPIRED DISCOVERY OF BIOFILM-ERADICATING AGENTS

Many cystic fibrosis (CF) patients suffer from initial S. aureus lung infections when they are young. As these patients age, P. aeruginosa coinfects their lungs and eradicates the established S. aureus infection using a series of phenazine antibiotics (70–73, Figure 16) with redox properties.^105–109 We reasoned the initial S. aureus infection to be biofilm-associated, as CF patients endure chronic and recurring lung infections throughout their life. Interested by the competitive microbial interaction between P. aeruginosa and S. aureus, our group set out to determine if phenazine antibiotic inspired molecules could be developed into bacterial biofilm-killing agents for clinical applications.

Figure 16.

Phenazine antibiotic inspired discoveries of new halogenated phenazine biofilm-eradicating agents. Our group has developed multiple synthetic pathways allowing us to establish a detailed structure-activity relationship profile for halogenated phenazine antibacterial agents. In addition, we have utilized RNA-seq technology to study the mode of action of HP-14 and define targets and pathways critical to bacterial biofilm viability using the WoPPER gene cluster analysis tool.

During initial studies, we synthesized a focused library of 13 phenazines (5 natural products, 8 synthetic derivatives; see 70–75; Figure 16) for biological evaluation. Results from the initial microbiological assays showed 2-bromo-1-hydroxyphenazine 74 to demonstrate the most potent antibacterial activities against S. aureus and S. epidermidis (MIC = 6.25 μM = 1.72 μMg/mL) among the naturally occurring phenazines in this collection, proving to be 8-fold more potent than pyocyanin 70 and >16- fold more potent than other P. aeruginosa-derived phenazines (71–73).¹¹⁰ Interestingly, 2-bromo-1-hydroxyphenazine was originally isolated from a marine Streptomyces strain,¹¹¹ and its synthetic analogue 2,4-dibromo-1-hydroxyphenazine 75 demonstrated the most potent antibacterial activities in the initial report (MIC = 1.56 μM = 0.55 μg/mL).¹¹⁰ We later determined that 75 is capable of eradicating established methicillin-resistant S. aureus biofilms by reporting a minimum biofilm eradication concentration (MBEC) of 100 μM (compared to vancomycin’s MBEC > 2000 μM under the same assay conditions).¹¹²

Following the identification of halogenated phenazine (HP) 75 as a biofilm-eradicating agent, we developed modular and complementary synthetic routes that have enabled our team to explore a diverse range of substituents at positions 6–9 of the phenazine heterocycle (see R₆-R₉ on structure 78; Figure 16).^113–116 We found this approach fruitful as several new HP analogues bearing diverse substitution patterns at positions R₆- R₈ (see 78) demonstrated significantly improved antibacterial and biofilm eradication activities, including HP-14 (76; MRSA, MIC = 0.3 μM, MBEC = 6.25 μM^112,113), when compared to parent HP 75.

Our most productive synthetic route to HP small molecules involves a palladium-catalyzed Buchwald-Hartwig coupling between diverse anilines 79 and nitroarene 80, which were then subjected to a reductive cyclization using sodium borohydride and sodium ethoxide to generate a series of 1- methoxyphenazines (81; Figure 16). The final synthetic sequence involves a boron tribromide (BBr₃) demethylation and bromination with N-bromosuccinimide (NBS) to yield target HP 82. This synthetic route led to the identification of HP 77, which demonstrates incredibly potent antibacterial (MIC = 0.1 μM = 0.04 μg/mL) and biofilm eradication (MBEC = 2.35 μM) activities against MRSA and other Gram-positive pathogens.

In other studies, HP-14 served as a probe to elucidate the mode of biofilm eradication using RNA-seq technology for transcript profiling in MRSA biofilms.¹¹⁷ This work began by treating established MRSA-1707 biofilms with low concentrations of HP-14 (1/10 MBEC, 0.625 μM) for 20 h before total RNA was extracted and subjected to RNA-seq (comparing RNA levels from HP-14 treated and vehicle treated MRSA biofilms), which revealed significant changes to 217 of 2738 gene transcripts (134 genes upregulated, 83 genes downregulated). A WoPPER analysis proved very useful in distilling the RNA-seq results of >200 individual transcripts down to 37 gene clusters up- or downregulated in response to HP-14 treatment (Figure 16).

Results from the WoPPER analysis clearly showed that HP-14 upregulates (“activated”) six gene clusters in MRSA biofilms that are involved in iron acquisition.¹¹⁷ These results aligned with our previous studies, which demonstrated that HPs operate through a metal(II)-dependent process and not a redox-based mechanism.^113–116 Active HP small molecules contain a metal chelating moiety that includes the oxygen atom of the 1-hydroxyl group and the adjacent nitrogen atom ofthe phenazine heterocycle, which form a stable 5-membered ring when bound to a metal cation.

Following the WoPPER analysis, we subjected one gene from each upregulated or downregulated gene cluster with known bacterial functions to real-time qPCR (RT-qPCR) experiments to validate the initial 20 h experimental results with HP-14 in MRSA biofilms.¹¹⁷ In addition to the six gene clusters involved in iron acquisition (isd, heme iron acquisition; sir/sbn, staphyloferrin B, siderophore; hts/sfa, staphyloferrin A, siderophore; MW0695, hypothetical protein similar to ferrichrome ABC transporters; fhuD1; ferric hydroxamate transporters; fhuBG, ferric hydroxamate transporter), initial RNA-seq results from several other gene clusters were confirmed/validated via RT-qPCR experiments (e.g., upregulated gene clusters: splB, serine proteases; oppF, oligopeptide transporters; downregulated gene clusters: gap, glycolysis; arcD, arginine deiminase; representative examples).

Following the initial confirmation of target MRSA biofilm genes at 20 h, we performed a time course study to see the initial changes in transcripts induced by HP-14. We tested each of the genes involved in iron uptake alongside select genes of interest (splB, oppF, gap) at 8 h and found four iron uptake genes to be significantly upregulated (isdB, sbnC, sfaA, MW0695) in addition to splB (serine proteases) and oppF (oligopeptide transporters).¹¹⁷ However, at 1 and 4 h, only the iron uptake gene transcripts were upregulated, demonstrating that isdB, sbnC, sfaA, and MW0695 are the initial genes that respond to HP-14 treatment. We were surprised that such low concentrations of HP-14 could induce a rapid response in established MRSA biofilms, which are notorious for being “dormant” bacterial populations. Interestingly, the metal-chelating agents EDTA and TPEN (a cell membrane permeable agent) did not activate iron uptake systems when tested alongside HP-14 and have not demonstrated biofilm eradication activities in our assays (MBEC > 2000 μM).

Our RNA-seq findings suggest the mode of action for HP-14 to eradicate MRSA biofilms is through the rapid induction of iron starvation,¹¹⁷ aligning with our previous findings that HPs can bind iron(ll) using UV-vis spectroscopy. It is well established that iron is a critical nutrient for bacteria to thrive; however, the iron(III) found in the environment is unable to diffuse through bacterial cell membranes.¹¹⁸ To overcome the challenge of acquiring iron from the environment, bacteria utilize sophisticated iron uptake systems that involve the biosynthesis and secretion of siderophore molecules into their surrounding environments. Siderophores are able to tightly bind iron(III), and bacteria have cell surface receptors that recognize and shuttle siderophore-iron(III) complexes into the cell. Once inside the bacterial cell, the reductive cytoplasm readily reduces iron(III) to iron(II), which results in a loss of affinity to iron by the siderophore and subsequent release of iron(II) for use in critical bacterial functions (e.g., respiration, DNA biosynthesis, etc.; Figure 17). Our results suggest that HP-14 (clog P 6.25) rapidly diffuses into MRSA biofilm cells and binds iron(II) that has been released by siderophores or heme.¹¹⁷ As a result, MRSA biofilm cells sense the stress of iron starvation and respond through the rapid transcription of iron uptake machinery in an attempt to offset HP-14’s mode of action. Our data also suggest that the general metal-chelating agents EDTA and TPEN do not have the same ability to penetrate MRSA biofilms, which is why no activation of iron uptake systems or biofilm eradication is observed when tested alongside halogenated phenazine small molecules.

Figure 17.

Proposed mechanism of MRSA-1707 biofilm eradication using HP-14 as a probe in RNA-seq investigations. A WoPPER gene cluster analysis was critical to this work by focusing >200 altered gene products in MRSA biofilms to ~30 cellular pathways for further investigations.

Current efforts are focused on developing new prodrug strategies to translate our halogenated phenazine agents through modification of the phenolic hydroxyl group. This strategy offers several advantages, which can be utilized to (1) modulate physicochemical properties (i.e., increasing water solubility), (2) enable bacterial-mediated cleavage of active HP structures following entry into a bacterial cell, and (3) prevent off-target metal chelation (potential deactivation of HP or toxicity liability; however, minimal cytotoxicity has been observed with HP analogues).^115,116 Advances from these efforts could provide ground-breaking treatment options for chronic and recurring bacterial infections that result from persistent biofilms.

CONCLUSIONS

Despite the significant challenges posed by antibiotic resistance and tolerance, we are encouraged to report several exciting advances in “chemical microbiology” inspired by Nature’s diverse inventory of molecules. This Review covers a unique series of innovative approaches aimed to address several problems currently associated with pathogenic bacteria. Collectively, these advances have combined synthetic organic chemistry, medicinal chemistry, microbiology, chemical biology, and animal studies in unique ways to pursue new and effective treatments against antibiotic-resistant and -tolerant infections.

ABBREVIATIONS

Ala

alanine

ATR

acid tolerance response

avg

average

BBr₃

boron tribromide

^tBu

tert-butyl

Bn

benzyl

Bpin/pinB

pinacol boronic ester group

CC₅₀

50% cytotoxic concentration

CcpA

carbon catabolite protein A

CIP

ciprofloxacin

CF

cystic fibrosis

CFU

colony forming unit (viable bacterial count)

DMF

dimethylformamide

DNA

deoxyribonucleic acid

DNM

deoxynybomycin

DTS

diverted total synthesis

EDTA

ethyl-enediaminetetraacetic acid

Et

ethyl

eq

equivalents

FDA

Food and Drug Administration

Fe

iron

FQR

fluoroquinolone resistant (bacteria)

HATU

hexafluorophosphate azabenzotria-zole tetramethyl uranium

HepG2

human liver cancer cell line

Hex

hexyl

HP

halogenated phenazine

IC₅₀

50% inhibitory concentration

Lac

lactate

LepB

SPase protein target in Gram-negative bacteria

Lys

lysine

MBEC

minimum biofilm eradication concentration

MDR

multidrug-resistant

Me

methyl

mg/kg

milligrams per kilogram

MIC

minimum inhibitory concentration (lowest test concentration resulting in the complete inhibition of bacterial growth)

mmol

millimole

MRSA

methicillin-resistant Staphylococcus aureus

MW

molecular weight

NaBH₄

sodium borohydride

NaOEt

sodium ethoxide

NBS

N-bromosuccinimide

NMR

nuclear magnetic resonance

PCR

polymerase chain reaction

Pd

palladium; Pen, pentyl; Ph, phenyl

PMB

para-methoxybenzyl (group)

Pr

propyl

RCM

ring-closing metathesis

RNA

ribonucleic acid

Ru

ruthenium

SAR

structure-activity relationship

SPase

type 1 signal peptidase (bacterial target)

TBAF

tetrabutylammonium fluoride

TBS

tert-butyldimethyl- silyl

Tf

triflate

μg/mL

micrograms per milliliter

TMS

trimethylsilyl

TPEN

N,N,N’,N’-tetrakis(2-pyridinylmethyl)- 1,2-ethanediamine

μM

micromolar

VRE

vancomycin-resistant enterococci

VRSA

vancomycin-resistant S. aureus

WT

wild-type

X-Phos

a phosphine ligand