Development of a Therapeutic Peptide for Cachexia Suggests a Platform Approach for Drug-like Peptides

Abstract

During the development of a melanocortin (MC) peptide drug to treat the condition of cachexia (a hypermetabolic state producing lean body mass wasting), we were confronted with the need for peptide transport across the blood–brain barrier (BBB): the MC-4 receptors (MC4Rs) for metabolic rate control are located in the hypothalamus, i.e., behind the BBB. Using the term “peptides with BBB transport”, we screened the medical literature like a peptide library. This revealed numerous “hits”—peptides with BBB transport and/or oral activity. We noted several features common to most peptides in this class, including a dipeptide sequence of nonpolar residues, primary structure cyclization (whole or partial), and a Pro-aromatic motif usually within the cyclized region. Based on this, we designed an MC4R antagonist peptide, TCMCB07, that successfully treated many forms of cachexia. As part of our pharmacokinetic characterization of TCMCB07, we discovered that hepatobiliary extraction from blood accounted for a majority of the circulating peptide’s excretion. Further screening of the literature revealed that TCMCB07 is a member of a long-forgotten peptide class, showing active transport by a multi-specific bile salt carrier. Bile salt transport peptides have predictable pharmacokinetics, including BBB transport, but rapid hepatic clearance inhibited their development as drugs. TCMCB07 shares the general characteristics of the bile salt peptide class but with a much longer half-life of hours, not minutes. A change in its C-terminal amino acid sequence slows hepatic clearance. This modification is transferable to other peptides in this class, suggesting a platform approach for producing drug-like peptides.

Cachexia-anorexia syndrome is a lean body mass (LBM) “wasting” condition associated with many chronic diseases, including malignancies and infectious diseases.¹⁻⁴ Thus, cachexia is not itself a disease but rather a condition associated with many diseases or conditions.⁵⁻¹⁰ The National Cancer Institute estimates that up to 40% of cancer deaths are directly due to cachexia.^11,12 Cachexia is characterized by anorexia and progressive loss of body weight (BW). Clinical evidence supports skeletal muscle depletion as a predictive factor in decreased overall survival.¹³ Energy expenditure in the presence of cachexia exceeds any attempt at supplemental caloric administration.¹⁴ While loss of skeletal muscle mass is the most obvious symptom, it is the largest single and most visible component of LBM; other effects are the true cause of cachexia-associated mortality, e.g., cachexia-induced high metabolic rate multi-organ failure.¹⁵⁻¹⁷

Pharmacological studies in our laboratory as well as others, in rats, and now dogs, confirm a relatively global effect of melanocortin (MC)-4 receptor (MC4R) antagonists in the reversal of many forms of cachexia.^12,18−21 Thus, the heterogeneity of different diseases is not germane to the induction or mitigation of cachexia. However, the overwhelming majority of MC4R antagonists that have been reported require intracerebroventricular (ICV) administration to produce their therapeutic effects.^8,12,22,23 Since parenteral administration is ineffective, this suggests their site of action is in the central nervous system (CNS), i.e., behind the blood–brain barrier (BBB), and not at circumventricular organs.²³

While the concept of a CNS origin for cachexia was historically surprising,^1,10,24 the multi-laboratory reports on the efficacy of ICV MC4R antagonists in alleviating many forms of cachexia could not be denied.^8,20 However, the development of an enteral or parenteral (i.e., more therapeutically acceptable than ICV) MC4R antagonist that would consistently alleviate cachexia has remained elusive.

One common link between all cachexia-inducing diseases is elevated systemic pro-inflammatory cytokines levels,^4,25−27 as well as tumor-associated factors,²⁸ acting on the CNS to produce elevated central cytokine levels²⁹ and activation of the CNS MC system.³⁰ Direct evidence that different pro-inflammatory cytokines could play a causal role in cachexia was shown by the temporary alleviation of experimental and clinical cachexia by administration of a specific anti-cytokine antibody.^31,32 Additional support for a role of cytokines in the etiology of cachexia comes from studies in which peripheral or central cytokine administration was able to produce a cachexia-like condition.³³⁻³⁵ Potential mechanisms for this are an increase in hypothalamic MC tone, by increasing the ratio of αMC to agouti-related protein (AGRP, an endogenous MC4R antagonist^36,37), as well as CNS inflammation.^37,38

The devastating clinical effects of cachexia were a major impetus for our attempt to develop an anti-cachexia peptide, i.e., one with drug-like activity, including BBB transport. This report describes the unique developmental approach and derivatizations used to develop our anti-cachexia peptide drug (TCMCB07), why these derivatizations may be applicable to other peptides, some of the unique pharmacokinetic (PK) properties of TCMCB07 (which are shared by other peptides of its transport class), and how nuclear magnetic resonance (NMR) spectroscopy of TCMCB07 and one of its analogs reveals important concepts in peptide secondary structure for the field of peptide active transport/drug development.

1. Results and Discussion

1.1. Development of a Drug-like MC-4 Receptor Antagonist

1.1.1. Background

Reports that a biologically active peptide under investigation had BBB transport and/or oral activity (OA) for unexplained reasons are scattered throughout the medical research literature.^22,39−44 The few attempts to explore this mechanism (or mechanisms) of action have generally failed to systematically define specific structural features governing these activities,⁴⁵⁻⁵⁰ i.e., a failure to produce transport activities by using candidate derivatizations. “Follow-on” chemical alterations to a drug-like peptide failed to enhance, and often lost, drug-like activity.⁴⁵ Despite these failures, searching for generalizable approaches to make peptides drug-like continues in a manner analogous to the “search for the holy grail”.⁵¹

Peptide libraries can be produced by synthesizing a large number of primary sequence-related peptides that can be examined for a specific activity,⁵² i.e., associating structure with function. As a new approach to the development of an anti-cachexia MC peptide drug, we collected the structures of a series of peptides from the medical literature expressing BBB transport and/or OA (see Table 1, peptides 1–5). These peptides formed an enriched (for transport activity) library that could be used for the recognition of common or similar structural features.

Table 1. Peptides Reported to Have a Form of Transport, Including Blood–Brain Barrier Transport, Oral Activity, or Liver-to-Bile Transport^a.

^aPeptides 1–5 were used for the initial production of MC antagonists with enhanced drug-like properties. Peptides 6–15 confirmed and extended the concept of a platform technology, providing potential examples of peptides with BBB transport. In the table, 14 of 15 structures are cyclized sequences (green shading of lowercase “c”). Underlined/shaded dipeptide sequences are commonly found in the listed peptides with transport. These motifs usually include at least one Pro-aromatic residue dipeptide (highlighted in blue) and at least one nonpolar dipeptide (mainly Pro-Val or Phe residues, highlighted in yellow). As described in section 1.3.2), a Pro adjacent (either side) to an aromatic motif stabilizes peptides into a β hairpin (found in 13 of 18 peptides listed), while the Pro/Phe/Val/Leu dipeptide is found in 17 of 18 peptides listed. Several of these peptides have multiple examples of these motifs. In 9 of the 18 peptides, one of the motifs is composed of the notational first and last residues of the depicted cyclized sequence, i.e., adjacent residues in the cyclized peptides. In some instances (peptides 6, 11–14), the two motifs overlap (e.g., Phe-Pro or Pro-Phe; see Results and Discussion). Notes on the sequences: ¹A subscript “c” before parentheses indicates a cyclized sequence within. ²Tle = tertiary leucine or 3-methylvaline. ³Nal = naphthylalanine. ⁴Shows both hepatic uptake and BBB transport but is degraded in liver.

We perceived common features in this library, which were distilled into a series of derivatizations and amino acid substitutions in a model MC4R antagonist peptide. We hypothesized that this would produce transport activity in the peptide. The result of this effort was a drug-like MC peptide, TCMCB07, with both parenteral and oral anti-cachexia activity in animal models.^53,54

The derivatizations used in TCMCB07 may serve as a model for how many different types of peptides can be endowed with drug-like properties, including OA, predictable PKs, long half-lives, and BBB transport.

1.1.2. A Platform Technology

Initial literature analysis showed that many peptides with reported BBB transport and/or OA had similar structural features (Table 1, peptides 1–5). These include cyclization, a proline residue(s) within or adjacent to the peptide’s pharmacophore, and a dipeptide sequence composed of nonpolar residues (in particular Pro/Phe/Leu-Val/Phe), often at the C-terminus.

Almost all previously described MC peptides with demonstrated in vitro efficacy as antagonists at the MC4R (e.g., SHU9119) were therapeutically effective in stimulating BW gain and food intake in rodent cachexia models only when given ICV; i.e., there was no effect with parenteral administration.^12,22 Previous reports clearly demonstrated that their critical target (metabolic-rate-regulating MC4Rs) was behind the BBB.¹² These data underscored the importance of discovering the critical peptide motif(s) for BBB transport.

We designed a series of MC antagonists by studying our peptide library (Table 1, peptides 1–5). This included an MC4R antagonist, PG932, which reportedly possessed parenteral stimulation of feeding activity; i.e., it crossed the BBB.²² Examining this library gave us insight into the requirements for transport in peptides.

We evaluated the presence of anti-cachexia activity in our peptides in a series of studies with a rat bacterial toxin-induced cachexia model. This assay utilized an intraperitoneal (IP) challenge with lipopolysaccharide (LPS) and an endotoxin from the cell membrane of Gram-negative bacteria and sequentially examined the anti-cachexia activity of each MC4R antagonist by ICV infusion (nmol doses), parenteral (subcutaneous; SC) administration (μmol doses), and oral gavage (3× the parenteral dose). Reversal of both anorexia and the loss of BW were used as evidence of treatment efficacy.

All of our analogs were effective anti-cachexia agents ICV, a few were also effective parenterally, and one parenterally effective analog was also effective when tested by oral gavage.

The relatively short duration of these cachexia screening studies precluded the evaluation of LBM gain by measuring skeletal muscle mass (e.g., in gastrocnemius muscle). However, longer term efficacy studies of TCMCB07 in experimental chronic kidney disease cachexia⁵³ showed a reversal of both BW and LBM loss after a 2 week administration period.

Initially, we evaluated a peptide designated as TCMCB01 (Ac-Nle-_c(Asp-Pro-DNal2′-Arg-Trp-Lys)-DThr-DPro-DThr-NH₂). While this peptide had a nonpolar, metabolism-resistant C-terminal tripeptide, it showed significant anti-cachexia activity only by ICV administration (data not shown).

We then chose PG932, a partially cyclized MC4R antagonist (Table 1, peptide 5), as a model structure to begin a sequential derivatization approach. Unlike “head-to-tail” cyclized peptides, partial cyclization divides the peptide’s primary structure into two distinct geographical regions, allowing individual targeting of amino acid residues and sequences in areas with different secondary structures. PG932, unlike a close derivative (SHU9119, see above), stimulates food intake by both parenteral and ICV administration.²² However, like SHU9119, PG932 has no parenteral anti-cachexia activity (no reversal of BW loss) against an LPS challenge.⁵³ This indicated that, while PG932 crosses the BBB, either its concentration at the MC4R is very low (poor systemic half-life or poor BBB transport) or it is a relatively weak in vivo MC4R antagonist that cannot overcome the cachexic effect of LPS.

PG932’s structure has two dipeptide features which differentiate it from the primary structure of SHU9119.²² These include a proline replacement for a histidine residue within the cyclized region of the primary structure (Table 1, peptide 5 sequence) and a linear C-terminus composed of a nonpolar dipeptide from the αMC C-terminal sequence, Pro-Val.⁶³ We sequentially, separately and then in combination, targeted derivatizations to these molecular areas in an attempt to dissect out the requirements for trans-cellular transport.

We first designed TCMCB02 (Ac-Nle-_c(Asp-Pro-DNal2′-Arg-Trp-Lys)-DPro-DVal-NH₂) as a peptide that would express parenteral, and possible oral, anti-cachexia activity. TCMCB02 had “d” enantiomers of proline and valine substituted in the C-terminal dipeptide of PG932 to enhance in vivo stability.⁶⁴ TCMCB02 had significant anti-cachexia activity when given by both ICV and parenteral routes of administration (Figure 1A,B) but had no detectable oral anti-cachexia activity. We then attempted to produce OA by substitution of other metabolism-resistant amino acids in the C-terminal dipeptide.

Figure 1.

Effects of MC4R antagonist analogs on food intake and body mass. Panels A and B show the effect of parenteral TCMCB02 (peptide) or saline (n = 6 for each group) on overnight food consumption and body weight following parenteral lipopolysaccharide (LPS) administration. Rats eat mainly at night, losing weight during the day and gaining it back at night when they feed. TCMCB02 stimulated feeding (panel A) compared to a baseline 4 day × 12 h dark cycle average. Saline control group rats ate 40% of their baseline, while TCMCB02-treated rats consumed 65%. Saline-treated rats gained little weight overnight (panel B), while TCMCB02-treated rats gained almost 5%. This approached a normal weight gain during their baseline period. Thus, TCMCB02 significantly ameliorated LPS-induced cachexia-anorexia. Panels C and D show the effect of saline or three different MC4R antagonists in rats given LPS to induce cachexia (n = 8 per group) on both food intake (panel C) and body weight (panel D, as percent of pre-LPS baseline). Measurements were made at 24 h after the LPS challenge, and drug candidates were given IP at 2 mg/kg. Saline controls showed anorexia and weight loss. TCMCB05 and TCMCB07 stimulated appetite and produced a significant weight gain. TCMCB06-administered rats ate significantly less food than those given the other drug candidates, with no weight gain. While the weight gain for food eaten with TCMCB05 and TCMCB07 appears less than that in panel B, the latter data were recorded at the end of the 12 h dark cycle when eating occurred. The data in panel D are 24 h data, which includes the dark/feeding phase and the subsequent 12 h of light cycle, when weight loss occurs. *p < 0.05, ***p < 0.001. Panels E and F show the effects of oral gavage of two parenterally active MC4R antagonists, TCMCB05 and TCMCB07, on food intake (panel E) and body weight (panel F) in rats given LPS to induce cachexia. Only TCB07 significantly ameliorated the anorexia and weight loss of LPS-induced cachexia. These data are 24 h data, including the dark/feeding phase and the subsequent 12 h of light cycle, when circadian weight loss occurs. *p < 0.05, ***p < 0.001.

In addition to d-amino acids, β-amino acid residues have also been shown to be metabolism-resistant⁶⁵ and are potentially superior to d-amino acids in maintaining biological activity.^65,66 We therefore designed TCMCB07 derivatives with C-terminal dipeptides composed of different β forms of proline and valine. Two examples of TCMCB02 analogs that we produced are Ac-Nle-_c[Asp-Pro-DNal(2′)-Arg-Trp-Lys]-βPro-β²Val-NH₂ (TCMCB05) and Ac-Nle-_c[Asp-Pro-DNal(2′)-Arg-Trp-Lys]-βPro-β³Val-NH₂ (TCMCB06). The difference between these two molecules is the shifting of either the valine α-carbon amino (TCMCB05) or carboxyl (TCMCB06) groups to the corresponding β-carbon. Only TCMCB05 showed parenteral anti-cachexia activity, compared to TCMCB06 or a saline control (Figure 1C,D). However, TCMCB05 did not produce significant therapeutic anti-cachexia activity when given orally (Figure 1E,F).

Substituting different forms of β-amino acid residues in the C-terminal dipeptide, with corresponding differences in BBB transport, demonstrated the highly specific structural requirements in the C-terminal dipeptide for parenteral anti-cachexia activity. That relatively subtle changes in a single amino acid residue of a nonapeptide prevented significant BBB transport suggests specific binding to a carrier.⁶⁷

We also synthesized TCMCB02 analogs with a residue change within the cyclized region. We substituted the prolyl residue with a histidyl, producing an analog designated as TCMCB03. This produced a cyclized sequence similar to that of MC4R antagonist SHU9119, but with the C-terminal dipeptide found in TCMCB02. Similar to SHU9119, TCMCB03 had anti-cachexic activity only after ICV administration.⁵³ These data indicated that derivatizations within the cyclized region of TCMCB02 or changes in its C-terminal dipeptide eliminated anti-cachexia activity, demonstrating that both regions of TCMCB02 contain dipeptide sequences necessary but individually insufficient for BBB transport activity.

While TCMCB02 was a potential anti-cachexia drug candidate, we examined an additional analog. The C-terminal dipeptide sequence of peptide 2 in Table 1 contains a valine analog, Tle (tertiary leucine or 3-methylvaline), in the penultimate position. This suggested the testing of a reversal of the C-terminal dipeptide in TCMCB02, DPro-DVal, to DVal-DPro (peptide designated as TCMCB07). This dipeptide sequence placed a DPro residue on the carboxyl side of a nonpolar residue, providing additional protection against C-terminal exopeptidases, since the (left-to-right) ultimate peptide bond would contain a secondary amine, i.e., in the imidic acid (proline) residue.⁶⁸ TCMCB07 (Ac-Nle-_c[Asp-Pro-DNal(2′)-Arg-Trp-Lys]-DVal-DPro-NH₂) had parenteral anti-cachexia activity (Figure 1C,D) and was subsequently tested and shown to have oral anti-cachexia activity (Figure 1E,F).

Our BBB transport studies of TCMCB07 were corroborated by collaborative studies with David Smith’s laboratory (University of Michigan). These studies demonstrated the specific binding and transport of TCMCB07 by OATP1A2, a bile salt solute carrier transporter, potentially responsible for TCMCB07’s BBB and gastrointestinal (GI) transport.⁶⁹ This solute transporter is in the right places (anatomically) and transports solutes in the right direction.

Our in vivo evidence for the GI transport of TCMCB07 has been confirmed by additional mass balance studies from the Smith laboratory.⁶⁹ The percent GI transport for TCMCB07 from our gavage studies (∼30%) is similar to that reported by Smith et al. Thus, TCMCB07 was designated as our lead anti-cachexia drug for further investigation.

1.1.3. TCMCB07: An MC4R Receptor Antagonist

TCMCB07 Binding Properties at Human, Dog, and Rat Melanocortin Receptors

In order to demonstrate the translational potential of TCMCB07 from our rat and canine cachexia efficacy data to human cachexia, we used this drug’s inhibition of rat and dog melanocortin receptors (MCRs) compared to its effects on human MCRs to demonstrate the potential trans-species efficacy of TCMCB07.

First, the binding properties of TCMCB07 in rat (r), dog (d), and human (h) MCRs were compared against [Nle⁴,DPhe⁷]-α-melanocyte-stimulating hormone (NDP-MSH), a non-specific super-potent analog of endogenous α-MSH.⁷⁰ NDP-MSH has been widely used in the functional studies of MCRs.⁷¹

TCMCB07 dose-dependently displaced ¹²⁵I-NDP-MSH binding at all MC1R, MC3R, MC4R, and MC5R of humans, dogs, and rats (Figure 2 and Table 2). Among hMCRs, hMC3R showed the lowest IC₅₀ (0.59 ± 0.08 nM), followed by hMC5R (8.07 ± 0.55 nM), hMC4R (13.58 ± 1.33 nM), and hMC1R (19.50 ± 2.35 nM). In rats, rMC3R displayed the lowest IC₅₀ (0.99 ± 0.10 nM), followed by rMC4R (2.58 ± 0.33 nM), rMC1R (9.31 ± 0.92 nM), and rMC5R (70.52 ± 7.05 nM). In dogs, dMC3R also showed the lowest IC₅₀ (1.46 ± 0.19 nM), followed by dMC4R (7.14 ± 1.99 nM), dMC5R (11.22 ± 2.51 nM), and dMC1R (53.64 ± 4.04 nM).

Figure 2.

Ligand-binding properties of MCRs in humans (A), rats (B), and dogs (C). HEK293T cells were transiently transfected with MC1R, MC3R, MC4R, or MC5R plasmids using a calcium phosphate precipitation method. Forty-eight hours after transfection, cells were incubated at 37 °C with DMEM/BSA containing 80 000 cpm of radiolabeled [¹²⁵I]-NDP-MSH without or with different concentrations of unlabeled 10^–11–10^–6 M TCMCB07. Data are expressed as percentage of the maximal binding value (B_max) from duplicate measurements within one experiment. The curves are representative of three independent experiments.

Table 2. Ligand-Binding Properties of MCRs in Humans, Rats, and Dogs.

	TCMCB07 binding IC50 (nM)a
	humans	rats	dogs
MC1R	19.50 ± 2.35	9.31 ± 0.92	53.64 ± 4.04
MC3R	0.59 ± 0.08	0.99 ± 0.10	1.46 ± 0.19
MC4R	13.58 ± 1.33	2.58 ± 0.33	7.14 ± 1.99
MC5R	8.07 ± 0.55	70.52 ± 7.05	11.22 ± 2.51

^aValues are expressed as mean ± SEM (n = 3).

TCMCB07 cAMP Signaling Properties at Human, Dog, and Rat MCRs

hMC1R and rMC1R showed high basal cyclic adenosine monophosphate (cAMP) production, and cMC1R had lower or no basal activity (Figure 3 and Table 3). TCMCB07 partially increased cAMP levels of hMC1R, dose-dependently increased intracellular cAMP generation of cMC1R (EC₅₀ = 3.00 ± 0.10 nM), and had no significant effect on cAMP levels of rMC1R (Figure 3 and Table 3).

Figure 3.

TCMCB07 and MC1R cAMP production in humans (A), rats (B), and dogs (C). HEK293T cells were plated into 6-well plates pre-coated with 0.1% gelatin. At approximately 70% confluency, cells were transiently transfected with hMC1R, rMC1R, or dMC1R plasmids using a calcium phosphate precipitation method. Forty-eight hours after transfection, intracellular cAMP production was determined by radioimmunoassay after stimulation with different concentrations of TCMCB07. Data are expressed as percentage of the maximal responses (R_max) from triplicate measurements within one experiment. All experiments were performed at least three times independently.

Table 3. Signaling Properties of MC1R and MC5R in Humans, Rats, and Dogs.

	TCMCB07-stimulated cAMP, EC50 (nM)a
	MC1R	MC5R
human	ND	1.42 ± 0.53
rat	ND	5.82 ± 1.56
dog	3.00 ± 0.10	0.0012 ± 0.0006

^aValues are expressed as mean ± SEM (n = 3).

To explore the potential effect of TCMCB07 on MC2Rs, cells were co-transfected with MC2R and MRAP1. Results showed that TCMCB07 exhibits different effects on three MC2Rs (Figure 4 and Table 4). TCMCB07 stimulated hMC2R with 55.97 ± 7.38% the R_max of ACTH(1–24). Similar EC₅₀ values between TCMCB07 and ACTH(1–24) were observed in hMC2R (Figure 4 and Table 4). In rats, B07 partially increased cAMP generation with 21.87 ± 3.94% the R_maxof ACTH(1–24). In contrast, TCMCB07 could not stimulate cAMP production of dMC2R (Figure 4 and Table 4).

Figure 4.

TCMCB07 and MC2R cAMP production in humans (A), rats (B), and dogs (C). HEK293T cells were co-transfected with MC2R and MRAP1 (1:1) (MRAP1a for human MC2R). cAMP production was determined by radioimmunoassay after stimulation with different concentrations of ACTH (1–24) or TCMCB07. ACTH (1–24) was used as a positive control. Data are expressed as percentage of the R_max of ACTH (1–24) from triplicate measurements within one experiment. All experiments were performed at least three times independently.

Table 4. Signaling Properties of Three MC2Rs.

	ligand-stimulated cAMP
	ACTH (1–24)		TCMCB07
	EC50 (nM)	Rmax (%)	EC50 (nM)	Rmax (%)
hMC2R + MRAP1a	1.90 ± 1.37	100	2.35 ± 1.50	55.97 ± 7.38
rMC2R + MRAP1	2.24 ± 0.20	100	2.55 ± 2.31	21.87 ± 3.94
dMC2R + MRAP1	3.43 ± 1.16	100	N/Aa	11.29 ± 1.54

^aCould not be determined.

To study the potential antagonistic effects of TCMCB07 on MC3Rs and MC4Rs, we examined their effects on NDP-MSH-stimulated cAMP generation in HEK293T cells transiently transfected with MC3R or MC4R of humans, dogs, and rats. Results showed that TCMCB07 causes a rightward shift of the NDP-MSH-induced cAMP response of three MC3Rs and three MC4Rs with increased EC₅₀ values and reduced R_max values (Figures 5 and 6, and Tables 5 and 6). hMC3R, rMC3R, and dMC3R showed high pA2 values of 9.46 ± 0.12, 9.39 ± 0.08, and 9.31 ± 0.15, respectively (Table 5). hMC4R, rMC4R, and dMC4R had pA2 values of 8.80 ± 0.06, 8.87 ± 0.06, and 8.66 ± 0.13, individually (Table 6).

Figure 5.

TCMCB07 and MC3R cAMP production in humans (A), rats (B), and dogs (C). HEK293T cells were transiently transfected with hMC3R, rMC3R, or dMC3R. Forty-eight hours after transfection, different concentrations of ligands were added into the media to make the final concentration of NDP-MSH ranging from 10^–12 to 10^–6 M, without or with the presence of 1, 10, or 100 nM TCMCB07. cAMP levels were determined by radioimmunoassay. Data are expressed as percentage of the R_max of the control group without TCMCB07 from triplicate measurements within one experiment. All experiments were performed at least three times independently.

Figure 6.

TCMCB07 and MC4R cAMP production in humans (A), rats (B), and dogs (C). HEK293T cells were transiently transfected with hMC4R, rMC4R, or dMC4R. Forty-eight hours after transfection, different concentrations of ligands were added into the media to make the final concentration of NDP-MSH ranging from 10^–12 to 10^–6 M, without or with the presence of 1, 10, or 100 nM TCMCB07. cAMP levels were determined by radioimmunoassay. Data are expressed as percentage of the R_max of the control group without TCMCB07 from triplicate measurements within one experiment. All experiments were performed at least three times independently.

Table 5. Effects of B07 on NDP-MSH-Stimulated Signaling of the Three MC3Rs^a.

		NDP-MSH	NDP-MSH + 1 nM TCMCB07	NDP-MSH + 10 nM TCMCB07	NDP-MSH + 100 nM TCMCB07	pA2
hMC3R	EC50 (nM)	0.35 ± 0.07	1.40 ± 0.16b	28.12 ± 1.17c	309.50 ± 37.91b	9.46 ± 0.12
	Rmax (%)	100	85.56 ± 10.04	95.59 ± 6 45	84.60 ± 10 12
rMC3R	EC50 (nM)	0.36 ± 0 03	1.40 ± 0.14c	39.22 ± 6.27b	561.57 ± 116.63b	9.39 ± 0.08
	Rmax (%)	100	96.07 ± 7.94	74.76 ± 8.20	65.47 ± 5.58b
dMC3R	EC50 (nM)	0.86 ± 0.08	1.67 ± 1.21	10.16 ± 0.75c	108.43 ± 32.08b	9.31 ± 0.15
	Rmax (%)	100	80.14 ± 3.90b	75.26 ± 2.36c	78.70 ± 6.37

^aValues are expressed as mean ± SEM (n = 3).

^bSignificant difference from the parameter of NDP-MSH, P < 0.05.

^cSignificant difference from the parameter of NDP-MSH, P < 0.01.

Table 6. Effects of B07 on NDP-MSH-Stimulated Signaling of the Three MC4Rs^a.

		NDP-MSH	NDP-MSH + 1 nM B07	NDP-MSH ± 10 nM B07	NDP-MSH ± 100 nM B07	pA2
hMC4R	EC50 (nM)	0.70 ± 0.18	1.07 ± 0.34	4.59 ± 0.80c	69.86 ± 26.77	8.80 ± 0.06
	Rmax (%)	100	90.30 ± 3.76	77.65 ± 16.00	56.42 ± 21.48
rMC4R	EC50 (nM)	0.20 ± 0.05	0.32 ± 0.11	9.90 ± 1.40b	371.11 ± 82.05b	8.87 ± 0.06
	Rmax (%)	100.00	95.26 ± 11.16	79.96 ± 4.89	70.90 ± 8.85
dMC4R	EC5o (nM)	0.33 ± 0.07	0.39 ± 0.10	5.22 ± 1.77	143.40 ± 56.25b	8.66 ± 0.13
	Rmax (%)	100	100.24 ± 8.33	94.72 ± 9.18	84.05 ± 3.93

^aValues are expressed as mean ± SEM (n = 3).

^bSignificant difference from the parameter of NDP-MSH, P < 0.05.

^cSignificant difference from the parameter of NDP-MSH, P < 0.01.

TCMCB07 could stimulate MC5Rs and dose-dependently increase intracellular cAMP generation (Figure 7 and Table 3). rMC5R showed the lowest EC₅₀ (0.0012 ± 0.0006 nM), followed by hMC5R (1.42 ± 0.53 nM) and dMC5R (5.82 ± 1.56 nM).

Figure 7.

TCMCB07 and MC5R cAMP production in humans (A), rats (B), and dogs (C). HEK293T cells were transiently transfected with hMC5R, rMC5R, or dMC5R plasmids. Forty-eight hours after transfection, cAMP production was determined by radioimmunoassay after stimulation with different concentrations of TCMCB07. Data are expressed as the percentage of the R_max from triplicate measurements within one experiment. All experiments were performed at least three times independently.

The above data are consistent with previously reported evidence that MC1R and MC5R have relatively few antagonists in MC.^72,73 This is unlike MC3R and MC4R, where sequential studies of the MC pharmacophore led to the use of DNal(2′) substitution for the histidyl residue in the MC pharmacophore, producing potent receptor antagonism in many MC analogs.⁷⁴⁻⁷⁶ Accordingly, TCMCB07, which contains DNal(2′), is an MC3R and MC4R antagonist but is an agonist for MC1R and MC5R (Figures 3, 5, and 6, and Tables 3, 4, and 5).

In terms of “off-target” side effects, we reported⁵⁴ increased pigmentation of hair (an MC1R effect) in dogs receiving TCMCB07 for weeks; no other side-effects (other than BW gain) were noted. The role of the MC5R in exocrine gland secretion was initially based on a lack of sebaceous gland secretion in MC5R knockout mice.⁷⁷ This led to the mapping of the MC5R to most, if not all, exocrine glands. While there is evidence of a role for the MC5R in epidermal exocrine glands (e.g., lacrimal, Harderian, preputial), the role of the MC5R in more physiologically important exocrine glands (e.g., GI tract) remains unproven.

Melanocortin receptor accessory protein 1 (MRAP1), a specific molecular chaperone, is essential for the expression of MC2R.^78,79 MC2R is specific for ACTH. Thus, to investigate the potential effect of TCMCB07 on MC2Rs, cells were co-transfected with MC2R and MRAP1. Our results demonstrated that, while TCMCB07 could increase cAMP levels of hMC2R and rMC2R, it has no effect on dMC2R (Figure 4 and Table 4). While the data suggest a potential effect of TCMCB07-mediated effect on adrenocortical hormone secretion, reports on the steroidogenic effects of αMSH and ACTH fragments containing the αMSH^4–10/ACTH^4–10 sequence in dispersed adrenal cells showed almost an 8 order of magnitude lower potency than ACTH.⁸⁰

Most MC peptides with a DNal(2′) residue are antagonists or have weak partial agonism for hMC3R/hMC4R.^74−76,81 This phenomenon also applies to TCMCB07, acting as antagonist on MC3R and MC4R of humans, rats, and dogs (Figures 5 and 6, and Tables 5 and 6). Additionally, pA2 is the estimated equilibrium dissociation constant for the antagonist, which is the dose of antagonist that requires a 2-fold increase in agonist concentration.⁸² A high pA2 value (>9.31 in three MC3Rs and >8.66 in three MC4Rs) indicated the high antagonist potency of TCMCB07 on MC3Rs and MC4Rs (Tables 4 and 5). Consistent with our in vitro experiments, TCMCB07 administration alleviates cachexia by increasing food intake and BW in rats with LPS-induced cachexia and anorexia,⁵³ similar to other anti-cachexia MCR antagonists.^81,83 Further, TCMCB07 reverses cachexia in the Lewis sarcoma model and experimentally induced chronic kidney disease,⁵³ and the effects of the drug can be reproduced in dogs.⁵⁴

In summary, TCMCB07 acted as an agonist or partial agonist at MC1Rs, MC2Rs, and MC5Rs. TCMCB07 was an antagonist for MC3Rs and MC4Rs of humans, rats, and dogs. TCMCB07 is a mixed MC3/4R antagonist, similar to other anti-cachexia MC antagonists,^84,85 and could theoretically have in vivo effects at both receptors. However, MC4R knockout mice resist the development of cachexia, while MC3R knockout mice show enhanced sensitivity.¹⁹ Thus, one might predict that blockade of both receptors (with similar IC₅₀ and percent inhibition at maximal concentration) might cancel out each receptor’s effect. This never occurs; MC3/4R antagonists are consistently anti-cachectic,^12,20,53,86 suggesting predominant MC4R inhibition in vivo.

Direct evidence that TCMCB07 inhibits hypothalamic MC4Rs in vivo is found in a recent report from Daniel Marks’ laboratory (Oregon Health Sciences University, Portland, OR), performed in collaboration with Tensive Controls’ scientists.⁵³ In two models of cachexia, a solid tumor cancer model (Lewis sarcoma) and a model of chronic kidney disease, hypothalamic AGRP gene expression was elevated. This is consistent with a compensatory (but relatively ineffective) elevation in AGRP gene expression to a tumor-induced increase in hypothalamic MC system activity.^12,53 TCMCB07 administration alleviated both forms of cachexia and also resulted in significant reductions in AGRP gene expression. This is an appropriate response to lowered MC tone via blockade of the MC4R.

1.2. TCMCB07: A Member of the Bile Salt Transporter Peptide Class

1.2.1. Background

During the pharmacological characterization of our lead anti-cachexia candidate, TCMCB07, we examined chromatograms of its plasma levels during PK experiments. We noted a late-eluting chromatographic peak (long after the main TCMCB07 peak had eluted) that seemed to be related to TCMCB07 plasma levels. This suggested an analogy to reports of late-eluting secondary drug peaks in PK studies, where the secondary peak was eventually identified as a drug that had undergone enterohepatic recycling.⁸⁷ This term describes a circulating drug that is extracted by the liver via a solute transporter, with secretion into bile followed by intestinal absorption back into the hepatic portal system. These data raised the possibility of the active transport of circulating TCMCB07 by the liver into bile.

This suggested “hepatic transport” as another literature search term to uncover additional drug-like peptides. To our surprise, we rediscovered a “forgotten” class of synthetic and natural peptides—some initially designed to be potential drugs—that were ligands for a multi-specific bile salt transporter (e.g., OATP1A2⁶⁹) located in many tissues, including the BBB (Table 1, peptides 6–15). While the structural similarities between these peptides and TCMCB07 were striking, the former had half-lives too short for therapeutic utility.^44,56,88 TCMCB07 has a half-life of hours,⁵⁴ not minutes, potentially due to relatively small differences in the peptide’s C-terminal sequence. In essence, our initial group of five peptides contributed to the design of an exemplary bile salt transport peptide before we even knew the class existed!

1.2.2. Primary Structure Analogies

Our fortuitous rediscovery of a peptide class that exhibits active transport through a multi-specific bile salt transport system (Table 1, peptides 6–15) led us to perform a side-by-side comparison of the primary structure of TCMCB07 with the first reported member of that class, cyclosomatostatin.⁴⁴

A comparison of the sequence of TCMCB07 to that of cyclosomatostatin revealed many sequence analogies (Figure 8). Both peptides contain prolyl residues adjacent to aromatic residues within their cyclized regions (see brackets). In addition, the head-to-tail cyclization in cyclosomatostatin (i.e., no amino or carboxy termini) produces a sequence of nonpolar residues, Phe⁵-DPro⁶-Phe¹, that are similar to both the Pro-DNal (Pro-aromatic residues) and DVal-DPro (nonpolar-Pro residues) in the cyclized and linear regions of TCMCB07. Thus, two peptide motifs potentially responsible for transport in TCMCB07 are contained within overlapping nonpolar dipeptides in cyclosomatostatin, suggesting these may be responsible for the transport effects noted for this peptide class in Table 1 (peptides 6–15).

Figure 8.

Sequence comparison of cyclosomatostatin and TCMCB07. In addition to the obvious amino acid residue or residue class similarities (arrows), both peptides contain a proline residue adjacent to an aromatic residue within their cyclized regions (parentheses), with potential implications for their secondary structure (see section 1.3).

Our production of drug-like MC4R antagonist peptides is a continuation of the peptide class first described by Ziegler et al.⁸⁹ They showed the ability of certain cyclic analogs of somatostatin to bind to and be transported by a hepatic multi-specific bile salt transporter(s). Since it was known that a multi-specific bile salt transporter was expressed in other tissues, they suggested that the derivatizations in the somatostatin structure which produced this form of active transport might be useful for producing other peptide drugs (i.e., a potential “platform technology”).

However, continued work on this peptide class (see Table 1, peptides 7–18) revealed the problem of an extremely efficient extraction rate of cyclosomatostatin from blood to bile, leading to a very poor systemic half-life.^{44,56,58,88,90,91} This problem, first described by Zeigler et al., was never thoroughly investigated, nor were the amino acid residues/motifs responsible for the bile salt-like transport ever defined.

So, while Ziegler et al. were correct in first suggesting that development of cyclosomatostatin could be used as a model/platform to impart similar transport activity to other peptides, the short in vivo half-life of all of these peptides made it a “platform to nowhere” (for an analogy see “bridge to nowhere” in California⁹²).

To define the structural basis for bile salt-like transport, we examined derivatizations in the two structurally defined regions of our MC4R antagonist peptides: within the partially cyclized sequence of TCMCB07, and in the linear C-terminal dipeptide extension. Previous work with bile salt transport peptides had mainly used head-to-tail cyclization (Table 1, peptides 6–14), with many dipeptide sequences conservatively replicated and overlapping within these peptides, making it difficult to discern which residues or sequences were critical for transport activity. The study of partially cyclized peptides, i.e., subdividing the primary structure into cyclized and linear areas (Table 1, peptides 4 and 5, and the MC4R antagonist peptides described in this report), eventually demonstrated a “necessary but insufficient” dipeptide sequence within each area. Only when both are present is therapeutically useful drug-like transport activity expressed.

The dipeptide (Pro-Nal; Nal = aromatic residue) within the TCMCB07 cyclized region is required for a hairpin secondary structure, critical for many forms of peptide trans-cellular transport (see section 1.3.2 and section 1.3.3). Conservative substitutions in the C-terminal dipeptide sequence (Val-Pro is one iteration) can be used to “fine-tune” the speed of transport (see section 1.1.1). For example, an innovative feature in our development of TCMCB07 was the reversal of the sequence in the C-terminal dipeptide of TCMCB02. This resulted in a bile salt transport peptide with a much longer half-life and OA.^53,54 This slower rate of hepatic extraction of TCMCB07 from blood into bile (see below) produced a “platform to somewhere”, i.e., to therapeutically useful drug-like activity.

1.2.3. Hepatic Uptake

To examine the temporal relationship between circulating TCMCB07 and its potential extraction into bile, we measured (by HPLC with fluorescence monitoring) plasma and bile levels of the peptide after a 3 mg/kg SC administration in rats. TCMCB07 levels in plasma and bile rose in a roughly parallel fashion, with the biliary levels lagging by approximately 30–60 min (Figure 9). After 150 min, plasma concentrations of the TCMCB07 began to fall, while the concentration of TCMCB07 in bile continued to increase. The hepatic uptake and biliary secretion of TCMCB07 are similar to those reported for peptides that are ligands for a multi-specific bile salt transporter (Table 1, peptides 6–12).

Figure 9.

Plasma and bile levels of TCMCB07 in rats following SC administration (3 mg/kg). The appearance of the TCMCB07 in blood precedes that in bile. The subsequent increases of TCMCB07 in these fluids are roughly parallel for the first 90 min, and then biliary TCMCB07 continues to increase as plasma levels fall. This probably reflects a lag in TCMCB07 extraction from blood vs secretion into bile.

1.2.4. Pharmacokinetics

Comparing SC vs IP administration of TCMCB07 produced a 6-fold greater excretion of the peptide in bile and urine over the first 3 h (Figure 10). This supports a “first pass” extraction effect, similar to that reported for other IP-delivered drugs that undergo hepatic secretion into bile.⁹³

Figure 10.

Comparison of excreted TCMCB07 after SC versus IP administration. SC administration of TCMCB07 produced a combined (bile + urine) 5% excretion of the administered dose over the first 3 h. In comparison, IP administration resulted in a 30% excretion of the administered dose: a 6-fold increase in excretory rate.

These differences in SC and IP extraction/excretion correlate with pharmacological responses. A collaborative report from Tensive Controls’ scientists and Zhu et al.⁵³ showed that SC administration of TCMCB07 in a rodent disease model produced anti-cachexia effects, specifically BW and LBM gain and enhanced food intake. In contrast, IP administration of the identical TCMCB07 dose only produced enhanced food intake. Thus, the pharmacological differences produced by comparing SC vs IP administration in the rat cancer model support different mechanisms for TCMCB07’s enhancement of food intake versus increases in BW and LBM.

1.2.5. Biodistribution and Excretion of Radiolabeled TCMCB07

A biodistribution study with SC ¹²⁵I-labeled TCMCB07 showed hepatic-to-bile transport into feces and renal clearance as the major excretory mechanisms for the drug (Figure 11). At 1 h post-injection, there was ∼50% retention of the injected dose (ID) of radiolabeled peptide pergram of skin and subcutaneous tissue in the injection site. At 30 min post-injection, the peptide levels in blood were ∼1.4% of the ID, and this level slowly increased to ∼2.1% over the next 5 h. Peak concentration in bile showed a correlation with maximal hepatic drug concentration at 60–75 min post-injection.

Figure 11.

Biodistribution over time of radiolabeled TCMCB07. SC ¹²⁵I-TCMCB07 in mice was initially concentrated within the injection site. Over time, radioactivity first appeared in the liver and gall bladder, and eventually intestine, and then feces. Small amounts appeared in other organ systems, most notably the kidney. A renal excretory mechanism was demonstrated by radioactivity in the cage paper (see text). Evidence that the radioactivity detected was not released from degraded peptide is shown in Figures 4 and 5, where intact TCMCB07 was measured by HPLC in bile as well as urine.

TCMCB07 was excreted by both hepatic and renal mechanisms, with the feces and cage paper (representing excreted urine) each containing about 50% of the ID by 48 h post-injection.

TCMCB07 radioactivity remains in the liver for hours as it is cleared into the gall bladder, then large intestine, and eventually feces. Importantly, these data do not show any significant accumulation of the peptide within a non-excretory organ or any hepatic retention (Figure 11, see 24 h post-injection hepatic radioactivity levels).

While all of our data, PK and biodistribution, suggest that SC TCMCB07 is eliminated from blood and excretory organs within 24 h, radioactivity continues to appear in feces during the subsequent 24 h (i.e., 48 h post-injection). This may be due to the delay in murine total GI transit time, 6–8 h.⁹⁴

1.2.6. Plasma Protein Binding of TCMCB07

Peptides are amphoteric compounds;⁹⁵ i.e., they have functional groups that can act as either an acid or a base, or they have a neutral charge. Other functional activities are also present, e.g., polarity and aromaticity (e.g., for cation−π interaction⁹⁶). This amphoteric nature allows peptides to bind to many other molecules, including metals, plastics, or other peptides and proteins. The data described in the next section for peptides that bind to cyclophilin as their mechanism of action for the regulation of cytokine gene expression exemplify the potential pharmacological importance of this ability. In addition, plasma protein binding can be an important factor contributing to the PK properties of a drug.

Previous studies of TCMCB07 in normal dogs⁵⁴ used SC doses of 0.75 or 2.25 mg/kg. This produced day 1 C_max values of 2.6 ± 0.3 and 4.9 ± 0.3 μg/mL, respectively. Thus, TCMCB07 concentrations utilized within the plasma protein binding study were 1.25, 2.5, and 5.0 μg/mL.

Peptide binding to plasma proteins was tested using either 100% canine plasma (CP) or H₂O + 0.01% pluronic acid (PA) as diluent/dialysate, with the peptide solution prepared in H₂O + 0.01% PA or 100% CP, respectively.

Calculation of the free fraction (f_u) of TCMCB07 was as follows:

The free fractions calculated for 5.0 μg/mL of TCMCB07 were 0.16 ± 0.01 and 0.17 ± 0.04 for peptide prepared in H₂O + 0.01% PA and 100% CP, respectively. This produced an average free fraction of 0.17 ± 0.03 for 5.0 μg/mL of TCMCB07 in CP. In comparison, the free fractions calculated for 2.5 μg/mL of TCMCB07 prepared in H₂O + 0.01% PA and 100% CP were 0.13 ± 0.01 and 0.14 ± 0.01, respectively. The average calculated free fraction of TCMCB07 for all samples was 0.15 ± 0.02.

These data indicate that >99% of TCMCB07 in the circulation is bound to plasma proteins, potentially contributing to an extension of the peptide’s half-life. In addition, the protein-binding abilities of TCMCB07 provide support for its potential binding to the intracellular protein cyclophilin as a mechanism for its regulation of CNS pro-inflammatory cytokine gene expression (next section).

1.2.7. Bile Salt Transporter Class and Pro-inflammatory Cytokines

As described above, we recently collaborated with Marks et al.⁵³ to examine the effects of TCMCB07 in several models of cachexia. Parenterally administered TCMCB07 suppressed CNS hypothalamic pro-inflammatory cytokine gene expression in a rodent cancer model.⁵³ This was somewhat surprising, since MC4R peptides have not been typically described as direct modulators of gene expression.⁹⁷ However, there are reports of cytokine gene immunomodulatory activity in peptides of the bile salt transporter class.⁹⁸⁻¹⁰¹

Table 1 includes several bile salt transporter class peptides with reported anti-inflammatory activity, such as antamanide (peptide 7) and cyclolinopeptide A (CLA; peptide 9). Both of these peptides inhibit pro-inflammatory cytokine gene expression^98,100 by preventing the transport of a transcription factor from the lymphocyte cytoplasm to the nucleus. For example, CLA binds to and activates a cytoplasmic protein, cyclophilin, which then binds to the cytoplasmic enzyme calcineurin, inhibiting its phosphorylating activity. The physiological role for calcineurin is regulation of cytokine gene expression through phosphorylation of an NF-AT family cytokine transcription protein, producing nuclear transport and consequential pro-inflammatory cytokine gene expression.¹⁰¹ CLA-dependent inhibition of NF-AT phosphorylation prevents translocation of this transcription factor into the nucleus, inhibiting cytokine gene activity. This mechanism of action is similar to that of cyclosporin, an immunoregulatory drug used in transplant patients.¹⁰¹

The presence of large numbers of cationic and aromatic residues in peptides of the bile salt transport class produce motifs that are emblematic of potential cation−π binding.⁹⁶ This non-covalent attraction between small molecules and proteins^96,102 is commonly found in ligand–protein binding, due to the high bond energy in cation−π interactions.⁹⁶ Thus, it is possible that these interactions mediate the binding of bile salt transport class peptides to intracellular proteins that regulate cytokine transcription factor activation. Preliminary evidence supporting this concept was presented, wherein a Pro-Phe-Phe motif (found in both antamanide and CLA) was shown to be predictive of a peptide’s binding to cyclophilin.⁹⁸

TCMCB07’s similarity in structure to the essential residues for immunomodulatory activity in bile salt transporter class, a Pro-Nal (an aromatic) motif, could be the basis of its cytokine gene regulatory activity.

1.3. Nuclear Magnetic Resonance Spectroscopy of TCMCB07

1.3.1. Background

Nuclear magnetic resonance (NMR) spectroscopy provides information about atomic details of molecular structure and motions of organic molecules, including polypeptides.¹⁰³ Proton (¹H) NMR, extended with NMR of ¹⁵N and ¹³C nuclei, is ideal for structural analysis of polypeptides.¹⁰⁴

1.3.2. Application to TCMCB07

Previous reports suggest enhanced transport of cyclized peptides.^45,50 However, an attempt to use this concept in a predictive fashion to produce drug-like properties in peptides failed.⁴⁵ To determine if the secondary structure of a bile salt transport class peptide is critical for its active transport, we used TCMCB07 as a model of this class and performed solution NMR structural studies. As a negative control for these experiments, we used an analog of TCMCB07 in which Pro³ was replaced by a histidyl residue, i.e., TCMCB03 (discussed in section 1.1.2). The MC antagonist pharmacophore in TCMCB03 (His-DNal-Arg-Trp) is identical to that in SHU9119. Both SHU9119 and TCMCB03 lack BBB transport and must be given ICV to produce anti-cachexia activity.^22,53 As discussed above, a prolyl residue within the cyclized region appears to be critical for BBB transport in peptides produced by our platform (see Table 1).

¹H NMR was used to compare and contrast the structural characteristics of TCMCB03 and TCMCB07. NMR spectra of TCMCB03 and TCMCB07 provided NOE-based distances, J-coupling information on dihedral angles, and temperature coefficients consistent with backbone amide groups in hydrogen bonds (see Supporting Information). The NOE-based close contacts and dihedral information were used to restrain molecular dynamics (MD) simulations¹⁰³ in both TCMCB03 and TCMCB07.

The NMR-restrained structural ensemble revealed that the cyclized region of TCMCB07 forms a hairpin with the Pro³ and DNal⁴ being central in the turn (Figure 12 and Supporting Information). The NMR-based structural model of TCMCB07 (Figure 12) suggests a hydrogen bond (see arrow) between Asp² (C=O) and Arg⁵ (N–H) that contributes to ring stabilization. The NMR results are consistent with the evidence that pairing of aromatic and proline residues contributes to peptide stabilization into a hairpin.^105,106

Figure 12.

Structural model of TCMCB07 simulated by molecular dynamics restrained by NMR measurements. The estimates of close contacts are based on NOEs and the torsion angle ranges upon J-couplings. This conformation agrees well with the NOEs and J-couplings, according to lowest violations of the restraints. The arrow marks a probable hydrogen bond (dashed line) between the Arg⁵ (N–H) and Asp² (C=O). This closes the hairpin centered at Pro³-DNal⁴ in the cyclized region of the molecule. See Supporting Information, Tables S1–S5 and Figure S1.

Though NOEs support a β turn in TCMCB03, ¹⁵N NMR (T₂) relaxation studies suggest the backbone of TCMCB03 is more flexible than that of TCMCB07 (see Supporting Information, Table S5). Thus, the His³ substitution for Pro³ in TCMCB03 appears to destabilize the TCMCB07 hairpin secondary structure. Further, the His³-induced changes in NMR peak positions are greatest between Arg⁵ through DVal⁸, consistent with the most disruption of the peptide structure occurring in this segment of the peptide sequence.

The covalently homogeneous TCMCB03 also possesses two sets of NMR peaks in both an aqueous and a deuterated dimethyl sulfoxide (DMSO-d₆) solution, suggesting two conformational populations in either solvent. The approximately 1:1 ratio of peak heights in the two populations suggests that the two sets of TCMCB03 conformers are similarly populated. Further, the equilibrium between the conformers did not shift within the range of temperature and pH changes tested. This is consistent with previously published evidence for multiple conformations in solution for SHU9119,¹⁰⁷ a structurally similar analog to TCMCB03 (as discussed above).

Thus, the His-for-Pro substitution in TCMCB03 has a global disruptive impact on its secondary structure and could be related to its absence of BBB transport. The more rigid hairpin secondary structure of TCMCB07 may underlie its transport activities. The overall importance of proline residues in peptide active transport, especially when paired with aromatic amino acid residues, is highlighted by the transport similarities between mammalian bile salt carrier peptides (e.g., Table 1, peptides 6–12) and certain cationic anti-microbial peptides (CAMPs; see next section).

1.3.3. Peptide Transport and Secondary Structure

Our NMR results underscore the potential importance of a hairpin secondary structure as a requirement for active transcellular transport in TCMCB07 and, by implication, other peptides in the bile salt transporter class. But how widespread is the hairpin requirement for active transport of peptides in general? There are many literature reviews of cyclic peptides having a greater incidence of transport properties, without definitive conclusions as to the basis for this effect.^45,46,49,50 We used the term “peptides and hairpin” to screen the literature to discover if a hairpin structure was predictive of active transport. Numerous hits from this peptide–hairpin library support the phylogenetic importance of a hairpin structure in transmembrane transport.

One or more proline residues in a peptide, especially when adjacent to an aromatic residue, produce hairpins, and both linear and cyclic proline-rich CAMPs show transport in procaryotes and eucaryotes.^108−110 This is probably due (at least in part) to the imino substituent in the proline ring, which restricts free rotation in a peptide bond.¹¹¹ For example, linear and cyclized proline-rich CAMPs show transport across the Gram-negative bacterial cell membrane by a non-lytic mechanism, mediated by the SbmA transporter.^{110,112−114}

Antamanide [Table 1, peptide 7; _c(VPPFFPPPFF)] is a fungal peptide with bile salt transporter activity, which has primary sequence analogies to an insect-derived proline-rich linear CAMP, oncocin (VDKPPYLPRPRPPRRIYNR-NH₂). This latter peptide is also transported across the bacterial cell membrane.¹⁰⁸ Comparing these two peptides, the bold shows paired prolyl residues, and the underlined dipeptides show groupings of prolyl and adjacent aromatic residues in each sequence. Further, many of these proline-rich CAMPs also possess mammalian BBB transport activity.^112,115,116

The hairpin structure requirement is not confined to proline-rich peptides. Invertebrates and plants have CAMPs with a cyclic structure, composed of disulfide bonds, which also form hairpins. These CAMPs also demonstrate membrane transport into both Gram-positive and Gram-negative bacterial species, as well as fungi.^117,118 An example of a CAMP with disulfide bonds that stabilize a hairpin secondary structure is the plant anti-fungal hairpin peptide, EcAMP1.¹¹⁷ EcAMP1 acts by binding to fungal cell membranes, followed by internalization and cytoplasmic accumulation for anti-fungal actions, without disturbance of cell membrane integrity.

Finally, there is both direct and indirect evidence that endogenous mammalian peptides with a stable hairpin structure (e.g., the NPY-PYY family^119,120) also show BBB transport.^121,122 Thus, synthetic peptide ligands of a multi-specific bile acid transporter have secondary structural requirements for trans-epithelial transport that are similar to those found in naturally occurring peptides with bacterial membrane and/or BBB transport activity. These latter peptides are present in bacteria, fungi, plants, and animals (insects through mammals).

2. Conclusions

We developed a drug-like MC4R antagonist peptide, TCMCB07, to treat cachexia-anorexia syndrome. TCMCB07 is orally active, has predictable PK, and can cross the BBB—a critical feature for an anti-cachexia drug. TCMCB07 is a member of a class of bile-salt-like actively transported peptides. However, unlike other peptides in this class, TCMCB07 has a half-life of hours, not minutes. The differences mediating hepatic extraction between TCMCB07 and other members of the bile salt transport class are subtle in the primary structure but profound in the production of drug-like properties.

The transport properties of TCMCB07 led us to perform solution NMR spectroscopy, where we compared TCMCB07 to a close analog, TCMCB03, that lacked transport properties. This revealed the potentially critical characteristic of a hairpin secondary structure for TCMCB07, compared to a more flexible, multiple-forms ring structure for TCMCB03, with multiple NMR peaks. The primary amino acid sequence mediating this effect was the Pro-for-His substitution, adjacent to an aromatic residue, in the MC pharmacophore of TCMCB07. In contrast, TCMCB03 contained the established MC pharmacophore, His-Phe-Arg-Trp.

Further literature analysis revealed that hairpin peptides represent a class imbued with different forms of transport properties. The ability of hairpin peptides, with anti-microbial actions mediated by transport across the bacterial membrane, to also cross the BBB demonstrates a universality of this structural requirement for peptide transport that hitherto was mainly unappreciated. Definitive evidence for this relationship is seen in mammalian peptides with hairpin structures, which also show BBB transport.

3. Experimental Procedures

3.1. Peptide Sequences

The non-commercially available peptide sequences used in these studies were designed by Tensive Controls’ scientists and synthesized by a solid-phase technique at the University of Missouri Peptide Synthesis Core, or under cGMP conditions by CPC, Inc. (Sunnyvale, CA).

3.2. Analytical Procedures

3.2.1. Plasma Extraction

Samples of plasma in a polypropylene microfuge had the majority of the plasma proteins precipitated with a 1:4 ratio of plasma to acetonitrile, followed immediately by vigorous vortexing for 30 s. Proteins were then pelleted, the supernatant was transferred to a new microfuge tube, and the acetonitrile was evaporated. Finally, the levels of peptide within each sample were analyzed via HPLC analysis. The percent recovery of either from rat plasma was >90%.

3.2.2. High-Performance Liquid Chromatography Assay

Following precipitation of proteins, plasma or bile samples were then diluted to between 400 and 500 μL using purified water. The extracts were separated on a HypersilGold C-18 column (4.6 mm i.d./25 cm length, 5 μm particle size, 17.5 nm pore size), which was eluted with a 15–50% acetonitrile/0.01% hydrochloric acid gradient. We used the unique fluorescence spectrum of TCMCB07’s naphthylalanine (Nal) aromatic residue¹²³ for post-column detection of eluting Nal-containing peptides. Florescence detection was with a Fluoat-01 Panorama spectrofluorometer (Arlington, VA) using settings of 229 nm excitation/337 nm emission.

3.2.3. Plasma Protein Binding Assay

We used a Rapid Equilibrium Dialysis (RED) plate (Thermo Fisher Scientific). This plate contains dialysis membranes with an 8000 Da molecular cutoff. The membranes are positioned between two wells in order to quantitate the diffusion and equilibrium of TCMCB07 between a TCMCB07-containing well and a well initially without the peptide.

TCMCB07 shows significant non-specific binding to the polystyrene RED plates. To reduce this binding, we pre-incubated plates with 12% canine plasma (CP) for 2.5 h at 37 °C, using a shaker incubator at 200 rpm. Following the incubation, wells were rinsed with distilled H₂O + 0.01% pluronic acid (PA). This was followed by the addition of TCMCB07 (1.25, 2.5, or 5.0 μg/mL) in H₂O + 0.01% PA or 100% CP to the interior (red ringed) well, while H₂O + 0.01% PA was added to the exterior (white ringed) well as the diluent. The plate was then sealed and incubated for 24 h at 37 °C in a shaker incubator at 340 rpm. At the end of the incubation, a 100 μL sample was removed from each well. Each sample was then prepared for HPLC by the addition of 400 μL of acetonitrile to precipitate proteins and centrifuged. The supernatant was used for HPLC analysis (see above).

3.2.4. Routes of Administration

Peptide or vehicle (0.9% saline) was given by SC or IP injection, or by oral gavage dissolved in deionized distilled water.

3.2.5. Pharmacological Assays/Models

All animal protocols were approved by the Institutional Animal Care and Use Committees of the University of Missouri–Columbia and Oregon Health Sciences University. Protocols were in compliance with the National Institutes of Health and U.S. Department of Agriculture Guidelines for Care and Use of Animals in Research.

Endotoxin Cachexia Model

Rats were maintained on a 12 h light/dark schedule with ad libitum access to food and water. Animals were handled daily for a minimum of three consecutive days to decrease non-specific handling stress and produce a consistent control food intake and BW measurement. On the day of the experiment, individually housed animals received IP injections of LPS dissolved in 0.5% low-endotoxin BSA, 0.9% saline, or 0.5% BSA in 0.9% saline and were placed in their home cage. Peptides were administered by either IP, SC, or oral gavage, depending on the type of study, 1 h after LPS administration. Relative cachexia was assessed by measurement of food intake and BW on the day of challenge and 1 day after.

Melanocortin Receptor Binding/Activation

Human ACTH(1–24) was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). [Nle⁴,DPhe⁷]-α-MSH (NDP-MSH) was purchased from Peptides International (Louisville, KY, USA). TCMCB07 was obtained from TCI Peptide Therapeutics (Columbia, MO, USA). All plasmids were subcloned into a pcDNA3.1 vector.

Human embryonic kidney (HEK) 293T cells (ATCC, Manassas, VA, USA) were cultured at 37 °C in a 5% CO₂-humidified atmosphere.¹²⁴ Cells were plated into 6-well plates precoated with 0.1% gelatin. At approximately 70% confluency, cells were transfected with 0.25 μg/μL MC1R, MC3R, MC4R, or MC5R using a calcium phosphate precipitation method.¹²⁵ For MC2R, cells were co-transfected with MC2R and MRAP1 (1:1) (MRAP1a for human MC2R).

The radioligand ligand-binding assay was described previously.^124,126 Forty-eight hours after transfection, cells were incubated at 37 °C with DMEM/BSA containing 80 000 cpm of radiolabeled [¹²⁵I]-NDP-MSH without or with different concentrations of unlabeled 10^–11–10^–6 M TCMCB07. Radioimmunoassay was used to determine cAMP production.^124,127 For MC2R, ACTH(1–24) was used as a positive control. To investigate effects of the antagonists of B07 on MC3R and MC4R, different concentrations of ligands were added into the media to make the final concentration of NDP-MSH ranging from 10 ^–12 to 10 ^–6 M, without or with the presence of 1, 10, or 100 nM B07.

The pA2 value was determined by Schild Plot analysis. All data are shown as mean ± SEM. GraphPad Prism 8.3 software (GraphPad, San Diego, CA, USA) was used to calculate the parameters of ligand binding and cAMP signaling assays.

Pharmacokinetic Studies

Studies were performed in isoflurane-anesthetized Sprague–Dawley rats with femoral vein catheters and (in some experiments) a common bile duct catheter.

3.6. NMR and Molecular Modeling

3.6.1. NMR

NMR experiments were carried out on an 800 MHz Bruker Avance III spectrometer with TCI cryoprobe at 300 K. 1D proton spectra were acquired between 280 and 320 K to measure the temperature coefficients of the amide protons. 2D NMR DQF-COSY, TOCSY, and NOESY spectra were recorded in all aqueous DMSO-d₆ solvents for assignment of peaks and NOEs (see Supporting Information for further details).

3.6.2. Molecular Modeling

The computational protocol for structural determination in solution consisted of restrained MD simulations, energy minimization, and cluster analysis (see Supporting Information for further details).

3.7. Statistics

Statistical analysis of daily food intake was assessed by two-way analysis of variance (ANOVA: time × treatment), with post-hoc analysis using a Bonferroni test. A one-way ANOVA followed by a post-hoc Bonferroni test was used to analyze cumulative food intake in experimental versus control groups. Cumulative food intake during TCMCB07 treatment was analyzed by Student’s t test. Significance was an effect at the p < 0.05 level.

The significance of differences in cAMP signaling parameters was determined in all cases by Student’s t test. Statistical significance was set at p < 0.05.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00270.

• Additional technical information on NMR studies and molecular modeling; Tables S1–S5 (coupling constants with dihedral angles or calculated side chain populations; temperature coefficients of amide protons of TCMCB07; backbone assignments of two conformers of TCMCB03; temperature coefficients of TCMCB03 vs TCMCB07; and temperature coefficient in DMSO-d₆ and ¹⁵N T₂ relaxation in H₂O for TCMCB03 and TCMCB07), Figure S1 (sequential NOEs between Nal⁶-Arg⁵ in both conformers of TCMCB03), and a lay summary (PDF)

Author Contributions

Participated in research design: Gruber, Newton Northup, Jiang, Tao, and Van Doren. Conducted experiments: Gruber, Newton Northup, Gallazzi, Jiang, and Ren-Lai. Contributed new reagents or analytical tools: Gruber and Tao. Performed data analysis: Gruber, Tao, Ren-Lai, Newton Northup, Gallazzi, Jiang, and Van Doren. Wrote or contributed to writing of manuscript: Gruber, Tao, Newton Northup, Gallazzi, Jiang, and Van Doren.

This work was supported by National Cancer Institute SBIR grants R43CA150703, R44CA150703, and R44CA210763 to K.A.G., and a U.S. Treasury Qualifying Therapeutic Discovery Project grant to Tensive Controls, Inc.

The authors declare the following competing financial interest(s): Dr. Kenneth Gruber is a corporate officer in Tensive Controls, Inc., and holds equity in the company.

Notes

Tensive Controls, Inc. will agree to a limited license, to allow the synthesis of milligram quantities of any peptide disclosed in this publication, for purposes of confirmation or other non-commercial uses.