Molecular design of peptide therapeutics via N-terminal modification.

Glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon are three naturally occurring peptide hormones that mediate glucoregulation. Several agonists representing appropriately modified native ligands have been developed to maximize metabolic benefits with reduced side-effects and many have entered the clinic as type 2 diabetes and obesity therapeutics. In this work, we describe strategies for improving the stability of the peptide ligands by making them refractory to dipeptidyl peptidase-4 catalyzed hydrolysis and inactivation. We describe a series of alkylations with variations in size, shape, charge, polarity, and stereochemistry that are able to engender full activity at the receptor(s) while simultaneously resisting enzyme-mediated degradation. Utilizing this strategy, we offer a novel method of modulating receptor activity and fine-tuning pharmacology without a change in peptide sequence.

Prolonged Activation of the GLP-1 Receptor via Covalent Capture.

The incretin gut hormone glucagon-like peptide-1 (GLP-1) has become a household name because of its ability to induce glucose-dependent insulin release with accompanying weight loss in patients. Indeed, derivatives of the peptide exert numerous pleiotropic actions that favorably affect other metabolic functions, and consequently, such compounds are being considered as treatments for a variety of ailments. The ability of native GLP-1 to function as a clinical drug is severely limited because of its short half-life in vivo. All of the beneficial effects of GLP-1 come from its agonism at the cognate receptor, GLP-1R. In our quest for long-lived activation of the receptor, we hypothesized that an agonist that had the ability to covalently cross-link with GLP-1R would prove useful. We here report the structure-guided design of peptide analogues containing an electrophilic warhead that could be covalently captured by a resident native nucleophile on the receptor. The compounds were evaluated using washout experiments, and resistance to such washing serves as an index of prolonged activation and covalent capture, which we use to tabulate longevity and robust long-lived GLP-1R agonism. The addition of SulF (cross-linkable warhead), an N-terminal trifluoroethyl group (for protease protection), and a C18 diacid lipid (protractor) all contributed to the increased wash resistance of GLP-1. The most effective compound based on the wash resistance metric, C2K26DAC18_K34SulF, has all three elements outlined and may serve as a blueprint and a proof-of-concept scaffold for the design of clinically useful molecules.

A Robust Platform for the Molecular Design of Potent, Protease-Stable, Long-Acting GIP Analogues.

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid peptide hormone that regulates postprandial glucose levels. GIP binds to its cognate receptor, GIPR, and mediates metabolic physiology by improved insulin sensitivity, ß-cell proliferation, increased energy consumption, and stimulated glucagon secretion. Dipeptidyl peptidase-4 (DPP4) catalyzes the rapid inactivation of GIP within 6 min in vivo. Here, we report a molecular platform for the design of GIP analogues that are refractory to DPP4 action and exhibit differential activation of the receptor, thus offering potentially hundreds of GIP-based compounds to fine-tune pharmacology. The lead compound from our studies, which harbored a combination of N-terminal alkylation and side-chain lipidation, was equipotent and retained full efficacy at GIPR as the native peptide, while being completely refractory toward DPP4, and was resistant to trypsin. The GIP analogue identified from these studies was further evaluated in vivo and is one of the longest-acting GIPR agonists to date.

A Non-Perturbative Molecular Grafting Strategy for Stable and Potent Therapeutic Peptide Ligands.

The gut-derived incretin hormone, glucagon-like peptide-1 (GLP1), plays an important physiological role in attenuating post-prandial blood glucose excursions in part by amplifying pancreatic insulin secretion. Native GLP1 is rapidly degraded by the serine protease, dipeptidyl peptidase-4 (DPP4); however, enzyme-resistant analogues of this 30-amino-acid peptide provide an effective therapy for type 2 diabetes (T2D) and can curb obesity via complementary functions in the brain. In addition to its medical relevance, the incretin system provides a fertile arena for exploring how to better separate agonist function at cognate receptors versus susceptibility of peptides to DPP4-induced degradation. We have discovered that novel chemical decorations can make GLP1 and its analogues completely DPP4 resistant while fully preserving GLP1 receptor activity. This strategy is also applicable to other therapeutic ligands, namely, glucose-dependent insulinotropic polypeptide (GIP), glucagon, and glucagon-like peptide-2 (GLP2), targeting the secretin family of receptors. The versatility of the approach offers hundreds of active compounds based on any template that target these receptors. These observations should allow for rapid optimization of pharmacological properties and because the appendages are in a position crucial to receptor stimulation, they proffer the possibility of conferring "biased" signaling and in turn minimizing side effects.

Mutation-Induced Functional Alterations of CCR6.

The Cys-Cys chemokine receptor 6 (CCR6) is a well-established modulator of inflammation. Although several genetic associations have been identified between CCR6 polymorphisms and immune system disorders (e.g., rheumatoid arthritis and Crohn's disease), the pharmacological effects of naturally occurring missense mutations in this receptor have yet to be characterized. In this study, we initially assessed G protein-mediated signaling and observed that wild-type (WT) CCR6 exhibited ligand-independent activity. In addition, we found that the five most frequent CCR6 missense variants (A89T, A150V, R155W, G345S, and A369V) exhibited decreased basal and/or ligand induced Gαi protein signaling. To complement the study of these loss-of-function variants, we engineered a set of constitutively active CCR6 receptors. Selected mutations enhanced basal G protein-mediated signaling up to 3-fold relative to the WT value. Using a bioluminescence resonance energy transfer assay we investigated the ability of each naturally occurring and engineered CCR6 receptor mutant to recruit ß-arrestin. In contrast to G protein-mediated signaling, ß-arrestin mobilization was largely unperturbed by the naturally occurring loss-of-function CCR6 variants. Elevated recruitment of ß-arrestin was observed in one of the engineered constitutively active mutants (T98P). Our results demonstrate that point mutations in CCR6 can result in either a gain or loss of receptor function. These observations underscore the need to explore how CCR6 natural variants may influence immune cell physiology and human disease.

Novel Probes Establish Mas-Related G Protein-Coupled Receptor X1 Variants as Receptors with Loss or Gain of Function.

The Mas-related G protein-coupled receptor X1 (MrgprX1) is a human seven transmembrane-domain protein with a putative role in nociception and pruritus. This receptor is expressed in dorsal root ganglion neurons and is activated by a variety of endogenous peptides, including bovine adrenal medulla peptide (BAM) and γ2-melanocyte-stimulating hormone (γ2-MSH). In the present work, we study how naturally occurring missense mutations alter the activity of MrgprX1. To characterize selected receptor variants, we initially used the endogenous peptide ligand BAM8-22. In addition, we generated and characterized a panel of novel recombinant and synthetic peptide ligands. Our studies identified a mutation in the second intracellular loop of MrgprX1, R131S, that causes a decrease in both ligand-mediated and constitutive signaling. Another mutation in this region, H133R, results in a gain of function phenotype reflected by an increase in ligand-mediated signaling. Using epitope-tagged variants, we determined that the alterations in basal and ligand-mediated signaling were not explained by changes in receptor expression levels. Our results demonstrate that naturally occurring mutations can alter the pharmacology of MrgprX1. This study provides a theoretical basis for exploring whether MrgprX1 variability underlies differences in somatosensation within human populations.

A general method for making peptide therapeutics resistant to serine protease degradation: application to dipeptidyl peptidase IV substrates.

Bioactive peptides have evolved to optimally fulfill specific biological functions, a fact which has long attracted attention for their use as therapeutic agents. While there have been some recent commercial successes fostered in part by advances in large-scale peptide synthesis, development of peptides as therapeutic agents has been significantly impeded by their inherent susceptibility to protease degradation in the bloodstream. Here we report that incorporation of specially designed amino acid analogues at the P1' position, directly C-terminal of the enzyme cleavage site, renders peptides, including glucagon-like peptide-1 (7-36) amide (GLP-1) and six other examples, highly resistant to serine protease degradation without significant alteration of their biological activity. We demonstrate the applicability of the method to a variety of proteases, including dipeptidyl peptidase IV (DPP IV), dipeptidyl peptidase 8 (DPP8), fibroblast activation protein α (FAPα), α-lytic protease (αLP), trypsin, and chymotrypsin. In summary, the "P1' modification" represents a simple, general, and highly adaptable method of generating enzymatically stable peptide-based therapeutics.

Membrane tethered bursicon constructs as heterodimeric modulators of the Drosophila G protein-coupled receptor rickets.

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, we have explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor rickets (rk). Rk/bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. We initially engineered two distinct MTL constructs, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or ß bursicon subunit. Coexpression of the two complementary bursicon MTLs triggered rk-mediated signaling in vitro. We were then able to generate functionally active bursicon MTLs in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-ß or ß-α. Carboxy-terminal deletion of 32 amino acids in the ß-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble bursicon. We have thus generated membrane-anchored bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of bursicon in the genetically tractable Drosophila model organism. In addition, our success in generating functionally diverse bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins.

Naturally occurring HCA1 missense mutations result in loss of function: potential impact on lipid deposition.

The hydroxy-carboxylic acid receptor (HCA1) is a G protein-coupled receptor that is highly expressed on adipocytes and considered a potential target for the treatment of dyslipidemia. In the current study, we investigated the pharmacological properties of naturally occurring variants in this receptor (H43Q, A110V, S172L, and D253H). After transient expression of these receptors into human embryonic kidney 293 cells, basal and ligand-induced signaling were assessed using luciferase reporter gene assays. The A110V, S172L, and D253 variants showed reduced basal activity; the S172L mutant displayed a decrease in potency to the endogenous ligand L-lactate. Both the S172L and D253H variants also showed impaired cell surface expression, which may in part explain the reduced activity of these receptors. The impact of a loss in HCA1 function on lipid accumulation was investigated in the adipocyte cell line, OP9. In these cells, endogenous HCA1 transcript levels rapidly increased and reached maximal levels 3 days after the addition of differentiation media. Knockdown of HCA1 using siRNA resulted in an increase in lipid accumulation as assessed by quantification of Nile Red staining and TLC analysis. Our data suggest that lipid homeostasis may be altered in carriers of selected HCA1 missense variants.

Discovery of dual-action membrane-anchored modulators of incretin receptors.

BACKGROUND: The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function. METHODOLOGY/PRINCIPAL FINDINGS: Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G(7)]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G(7) into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G(7)]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes. CONCLUSIONS: These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target.

Two naturally occurring mutations in the type 1 melanin-concentrating hormone receptor abolish agonist-induced signaling.

The melanin-concentrating hormone (MCH) receptor type 1 (MCHR1) is a seven-transmembrane domain protein that modulates orexigenic activity of MCH, the corresponding endogenous peptide agonist. MCH antagonists are being explored as a potential treatment for obesity. In the current study, we examined the pharmacological impact of 11 naturally occurring mutations in the human MCHR1. Wild-type and mutant receptors were transiently expressed in human embryonic kidney 293 cells. MCHR1-mediated, Gα(i)-dependent signaling was monitored by using luciferase reporter gene assays. Two mutants, R210H and P377S, failed to respond to MCH. Five other variants showed significant alterations in MCH efficacy, ranging from 44 to 142% of the wild-type value. At each of the MCH-responsive mutants, agonist potency and inhibition by (S)-methyl 3-((3-(4-(3-acetamidophenyl)piperidin-1-yl)propyl)carbamoyl)-4-(3,4-difluorophenyl)-6-(methoxymethyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (SNAP-7941), an established MCHR1 small-molecule antagonist, were similar to wild type. To explore the basis for inactivity of the R210H and P377S mutants, we examined expression levels of these receptors. Assessment by enzyme-linked immunosorbent assay revealed that cell surface expression of both nonfunctional receptors was comparable with wild type. Overnight treatment with SNAP-7941, followed by washout of antagonist, enhanced MCH induced signaling by the wild-type receptor and restored MCH responsiveness of the P377S but not the R210H variant. It is of note that the two loss-of-function mutants were identified in markedly underweight individuals, raising the possibility that a lean phenotype may be linked to deficient MCHR1 signaling. Formal association studies with larger cohorts are needed to explore the extent to which signaling-deficient MCHR1 variants influence the maintenance of body weight.

The µ-opioid receptor variant N190K is unresponsive to peptide agonists yet can be rescued by small-molecule drugs.

The µ-opioid receptor (MOR) plays an important role in modulating analgesia, feeding behavior, and a range of autonomic functions. In the current study, we investigated the degree to which 13 naturally occurring missense mutations affect the pharmacological properties of the human MOR. After expression of each receptor in human embryonic kidney 293 cells, signaling (Gα(i/o)-mediated) induced by peptide agonists was assessed using luciferase reporter gene assays. Multiple mutants (S66F, S147C, R260H, R265C, R265H, and S268P) show a significant reduction in agonist potency. At the N190K variant, agonist-mediated signaling was essentially absent. Enzyme-linked immunosorbent assay, microscopic analysis, and radioligand binding assays revealed that this mutant shows markedly reduced cell-surface expression, whereas all other receptor variants were expressed at normal levels. Surface expression of the N190K variant could be increased by incubation with the alkaloid agonist buprenorphine or with either naltrexone or naloxone, structurally related MOR antagonists. We were surprised to find that both putative antagonists, despite being inactive at the wild-type MOR, triggered a concentration-dependent increase in N190K receptor-mediated signaling. In contrast, peptidic ligands failed to promote expression or rescue function of the N190K mutant. Subsequent analysis of the N190K variant in an ethnically diverse cohort identified this isoform in a subgroup of African Americans. Taken together, our studies reveal that the N190K mutation leads to severe functional alterations and, in parallel, changes the response to established MOR ligands. The extent to which this mutation results in physiological abnormalities or affects drug sensitivity in selected populations (e.g., those with chronic pain or addiction) remains to be investigated.

TGF-beta regulates T-cell neurokinin-1 receptor internalization and function.

Substance P (SP) is a proinflammatory mediator implicated in inflammatory bowel disease (IBD) and other inflammatory states. SP acts by stimulating the neurokinin-1 receptor (NK-1R) on T lymphocytes and other cell types, and regulates these cells in a complex interplay with multiple cytokines. The mechanisms of interaction among these inflammatory mediators are not yet fully understood. Here, we demonstrate that function of the NK-1R, a member of the G protein-coupled receptor (GPCR) superfamily, is modulated by TGF-beta. The latter acts not on a GPCR but via serine-threonine kinase-class receptors. By flow confocal image analysis, we demonstrate that TGF-beta delays SP-induced NK-1R internalization on mucosal T cells isolated from a mouse model of IBD and on granuloma T cells in murine schistosomiasis. Furthermore, luciferase reporter-gene assays revealed that NK-1R stimulation activates the nuclear factor of activated T cell- and activator protein-1-dependent signaling pathways, which are known triggers of effector T-cell cytokine production. TGF-beta markedly increases SP-induced activation of these signaling cascades, suggesting that delayed NK-1R internalization results in enhanced signaling. Providing a link to amplified immune function, SP and TGF-beta, when applied in combination, trigger a strong release of the proinflammatory cytokines IFN-gamma and IL17 from intestinal inflammatory T cells, whereas either agonist alone shows no effect. These observations establish precedent that members of two distinct receptor superfamilies can interact via a previously unrecognized mechanism, and reveal a paradigm of GPCR transregulation that is relevant to IBD and possibly other disease processes.

Pharmacological characterization of human incretin receptor missense variants.

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (>1000 and 25- fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated.

Neurogenin 3-specific dipeptidyl peptidase-2 deficiency causes impaired glucose tolerance, insulin resistance, and visceral obesity.

The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. Here we introduce the N-terminal dipeptidase, DPP2, as a novel regulator of the glucose metabolism. We generated mice with a neurogenin 3 (NGN3)-specific DPP2 knockdown (kd) to explore a possible role of DPP2 in maintaining metabolic homeostasis. These mice spontaneously developed hyperinsulinemia, glucose intolerance, and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in DPP2 kd mice, which was associated with a significant increase in adipose tissue mass and enhanced liver steatosis but no difference in body weight. In accordance with these findings, the mutant mice had a higher rate of respiratory exchange than the control littermates. This phenotype was exacerbated with age and when challenged with a high-fat diet. We report, for the first time, that DPP2 enzyme activity is essential for preventing hyperinsulinemia and maintaining glucose homeostasis. Interestingly, the phenotype of NGN3-DPP2 kd mice is opposite that of DPP4 knockout mice with regard to glucose metabolism, namely the former have normal glucagon-like peptide 1 levels but present with glucose intolerance, whereas the latter have increased glucagon-like peptide 1, which is accompanied by augmented glucose tolerance.

Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

Class B1 (secretin family) G protein-coupled receptors (GPCRs) modulate a wide range of physiological functions, including glucose homeostasis, feeding behavior, fat deposition, bone remodeling, and vascular contractility. Endogenous peptide ligands for these GPCRs are of intermediate length (27-44 aa) and include receptor affinity (C-terminal) as well as receptor activation (N-terminal) domains. We have developed a technology in which a peptide ligand tethered to the cell membrane selectively modulates corresponding class B1 GPCR-mediated signaling. The engineered cDNA constructs encode a single protein composed of (i) a transmembrane domain (TMD) with an intracellular C terminus, (ii) a poly(asparagine-glycine) linker extending from the TMD into the extracellular space, and (iii) a class B1 receptor ligand positioned at the N terminus. We demonstrate that membrane-tethered peptides, like corresponding soluble ligands, trigger dose-dependent receptor activation. The broad applicability of this approach is illustrated by experiments using tethered versions of 7 mammalian endogenous class B1 GPCR agonists. In parallel, we carried out mutational studies focused primarily on incretin ligands of the glucagon-like peptide-1 receptor. These experiments suggest that tethered ligand activity is conferred in large part by the N-terminal domain of the peptide hormone. Follow-up studies revealed that interconversion of tethered agonists and antagonists can be achieved with the introduction of selected point mutations. Such complementary receptor modulators provide important new tools for probing receptor structure-function relationships as well as for future studies aimed at dissecting the tissue-specific biological role of a GPCR in vivo (e.g., in the brain vs. in the periphery).

Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1.

The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis, and characterization of fluorinated analogues of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogues displayed higher proteolytic stability with simultaneous retention of biological activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions.