Non-peptide Analogues Research Articles

Synthesis and enzyme inhibitory activities of novel peptide isosteres.

Design and synthesis of metabolically stable peptide analogs that can either mimic or block the bioactivity of natural peptides or enzymes is an important constituent of bioorganic and medicinal chemistry research. Isosteric replacement of a scissile peptide bond represents a viable and popular approach in the rational design of peptidomimetics. Peptidomimetics find applications as drugs, in protein engineering and so on. This is evident from the wealth of therapeutically useful peptidomimetic leads incorporating any of the peptide isosteres that are currently available. In this review, we have given a brief account of the types of peptide isosteres widely known till date. With this background, we have described some of the recent developments in synthetic approaches. This includes methods involving a common intermediate to synthesize different possible isosteres and their peptide analogs, solid phase synthesis and combinatorial approach. One such method involving stereoselective nitrile oxide cycloaddition as the key step has been studied extensively in our research laboratory. Finally, we have also discussed about some of the recent reports on the design and inhibitory activities of peptidic or non-peptidic analogs against aspartic proteases (HIV-1, renin, ACE and pepsin) and peptide analogs of an immunomodulating hexapeptide.

Unique overlap in the prerequisites for thrombin inhibition and oral bioavailability resulting in potent oral antithrombotics.

Despite intense research over the last 10 years, aided by the availability of X-ray structures of enzyme-inhibitor complexes, only very few truly orally active thrombin inhibitors have been found. We conducted a comprehensive study starting with peptide transition state analogues (TSA). Both hydrophobic nonpeptide analogues as well as hydrophilic peptidic analogues were synthesized. The bioavailability in rats and dogs could be drastically altered depending on the overall charge distribution in the molecule. Compound 27, a tripeptide TSA inhibitor of thrombin, showed an oral bioavailability of 32% in rats and 71% in dogs, elimination half-lives being 58 and 108 min, respectively. The thrombin inhibition constant of compound 27 was 1.1 nM, and in an in vivo arterial flow model, the ED(50) was 5.4 nmol/kg.min, comparable to known non-TSA inhibitors. A molecular design was found that combines antithrombotic efficiency with oral bioavailability at low dosages.

Inhibitors of leukotriene A4 (LTA4) hydrolase as potential anti-inflammatory agents.

Leukotriene A4 (LTA4) hydrolase is a zinc-containing enzyme which stereospecifically catalyzes the hydrolysis of the epoxide LTA4 to the diol leukotriene B4 (LTB4). There is substantial evidence that LTB4 plays a significant role in the amplification of many inflammatory disease states. Therapeutic agents which selectively inhibit LTA4 hydrolase would block the formation of LTB4 and thus be potentially useful for the treatment of inflammation. Numerous inhibitors of LTA4 hydrolase have been reported over the past 15-20 years. Several early inhibitors were based on the structure of the natural substrate, LTA4. Later approaches utilized known inhibitors of related zinc-containing metalloproteinases and led to the identification of captopril, bestatin and kelatorphan as potent inhibitors of LTA4 hydrolase. This led to the design of a number of peptide and non-peptide analogs which contained potential zinc-chelating moieties, including thiols, hydroxamates and norstatines. A more recent series of non-peptidic, non-zinc chelating inhibitors of LTA4 hydrolase has been reported. This work led to the identification of several novel classes of analogs, including imidazopyridines, amidines and cyclic and acyclic amino acid derivatives, ultimately resulting in the identification of two potential clinical candidates SC-56938 and SC-57461A.

Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B Receptor Is Critical for the High Selectivity of the Peptoid Antagonist PD168368

Peptoid antagonists are increasingly being described for G protein-coupled receptors; however, little is known about the molecular basis of their binding. Recently, the peptoid PD168368 was found to be a potent selective neuromedin B receptor (NMBR) antagonist. To investigate the molecular basis for its selectivity for the NMBR over the closely related receptor for gastrin-releasing peptide (GRPR), we used a chimeric receptor approach and a site-directed mutagenesis approach. Mutated receptors were transiently expressed in Balb 3T3. The extracellular domains of the NMBR were not important for the selectivity of PD168368. However, substitution of the 5th upper transmembrane domain (uTM5) of the NMBR by the comparable GRPR domains decreased the affinity 16-fold. When the reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids that differed between NMBR and GRPR in the uTM5 region were exchanged, but only the substitution of Phe(220) for Tyr in the NMBR caused a decrease in affinity. When the reverse study was performed to attempt to demonstrate a gain of affinity in the GRPR, the substitution of Tyr(219) for Phe caused an increase in affinity. These results suggest that the hydroxyl group of Tyr(220) in uTM5 of NMBR plays a critical role for high selectivity of PD168368 for NMBR over GRPR. Receptor and ligand modeling suggests that the hydroxyl of the Tyr(220) interacts with nitrophenyl group of PD168368 likely primarily by hydrogen bonding. This result shows the selectivity of the peptoid PD168368, similar to that reported for numerous non-peptide analogues with other G protein-coupled receptors, is primarily dependent on interaction with transmembrane amino acids.

NMR studies of active N-terminal peptides of stromal cell-derived factor-1. Structural basis for receptor binding.

Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine family, is the only chemokine to bind to the receptor CXCR4. This receptor is also a co-receptor for syncytia-inducing forms of HIV in CD4(+) cells. In addition, SDF-1 is responsible for attracting mature lymphocytes to the bone marrow and can therefore contribute to host versus graft rejection in bone marrow transplantation. Clearly, by manipulating SDF-1 activity, we could find a possible anti-viral AIDS treatment and aid in bone marrow transplantation. SDF-1 binds to CXCR4 primarily via the N terminus, which appears flexible in the recently determined three-dimensional structure of SDF-1. Strikingly, short N-terminal SDF-1 peptides have been shown to have significant SDF-1 activity. By using NMR, we have determined the major conformation of the N terminus of SDF-1 in a 17-mer (residues 1-17 of SDF-1) and a 9-mer dimer (residues 1-9 of SDF-1 linked by a disulfide bond at residue 9). Residues 5-8 and 11-14 form similar structures that can be characterized as a beta-turn of the beta-alphaR type. These structural motifs are likely to be interconverting with other states, but the major conformation may be important for recognition in receptor binding. These results suggest for the first time that there may be a link between structuring of short N-terminal chemokine peptides and their ability to activate their receptor. These studies will act as a starting point for synthesizing non-peptide analogs that act as CXCR4 antagonists.

The effect of growth hormone secretagogues and neuropeptide Y on hypothalamic hormone release from acute rat hypothalamic explants.

Growth hormone (GH) secretagogues (GH-releasing peptides and their non-peptide analogues) stimulate growth hormone release via specific G-protein coupled receptors both directly from the pituitary gland and through stimulation of the hypothalamus. The exact mechanism of action in the hypothalamus is not known. The presence of endogenous GH releasing hormone (GHRH) seems to be necessary for the in-vivo actions of growth hormone secretagogues (GHSs), but data suggest that further factors must be involved as well. The effect of GHSs is not entirely specific for the GH axis; they release prolactin and stimulate the hypothalamo-pituitary-adrenal axis causing elevations in circulating ACTH and cortisol levels in both animal and human studies. Recently, it has also been suggested that GHSs stimulate hypothalamic neuropeptide Y (NPY) neurones. In the present study, we have therefore investigated the direct effect of several GHSs (GHRP-6, hexarelin and the non-peptide analogues L-692, 429 and L-692, 585) on GHRH, somatostatin (SS), corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) release in vitro in an acute rat hypothalamic incubation system. We also assessed the effect of NPY on GHRH, SS and AVP release. Freshly removed hypothalami were incubated in control media for 20 min and then in 1-4 consecutive 20-min periods in each of the test substances at different concentrations. There was no significant change in either the basal or potassium-stimulated release of GHRH or SS at low concentrations of any of the secretagogues; however, at millimolar doses a paradoxical inhibition of GHRH was observed with GHRP-6, hexarelin and L-692 585 (data are expressed as the ratio of treated to preceding basal release; at 20 min control group: 0.97+/-0.02, GHRP-6: 0.55+/-0.04, P<0.001 compared to control group; hexarelin: 0. 56+/-0.06, P<0.001, L-692,585: 0.70+/-0.03, P<0.001), while SS was stimulated after 60 or 80 min (at 80 min control: 0.80+/-0.03, hexarelin: 1.23+/-0.07, P<0.05 and L-692,585: 1.37+/-0.11, P<0.05). GHSs stimulated hypothalamic AVP release (at 20 min control: 0. 99+/-0.06 ratio to basal release, 10-4 M concentration of GHRP-6: 6. 31+/-1, P<0.001, hexarelin: 1.88+/-0.4, P<0.01, L-692,429: 1.90+/-0. 5, P<0.05 and L-692,585: 2.34+/-0.96, P<0.01), while no stimulatory effect was found on CRH release. NPY significantly stimulated SS and inhibited basal and potassium-stimulated GHRH release, while potentiating potassium-evoked AVP secretion. The Y1 receptor antagonist BIBP 3226 did not inhibit the effects of NPY on SS, GHRH or AVP release. We therefore conclude that, in this in-vitro rat hypothalamic incubation model, growth hormone secretagogues stimulate the release of AVP but have no effect on either GHRH, SS or CRH at low doses; at high doses paradoxically they inhibit the hypothalamic GH axis similar to in-vivo data in the rat. We speculate that these effects might be mediated by NPY.

Design criteria for molecular mimics of fragments of the beta-turn. 1. C alpha atom analysis.

Peptides represent an extensive class of biologically active molecules. They may be used as leads in the development of novel therapeutic agents provided the pharmacophoric information present within them can be translated into non-peptide analogs that lack the peptide backbone and are stable to proteolysis. This is the rationale for peptidomimetic drug design. Frequently, the beta-turn has been implicated as a conformation important for biological recognition of peptides. Empirical evidence from known peptidomimetics, coupled with a theoretical model of peptide binding and the observation that glycine and proline residues are common within the beta-turn, has suggested the design of molecules to mimic placement of between two and four of the side-chains. The moderate number of different beta-turn conformations, combined with the combinatoric nature of side-chain selection complicates the procedure. In this paper, cluster analysis has been used to classify the arrangement of C alpha atoms about the various fragments of the beta-turn. Recombination of the observed patterns provides a general model for the beta-turn which may be used as an effective screen for potential peptidomimetic scaffolds in chemical databases.

Somatostatin Receptor Ligands and Their Use in the Treatment of Endocrine Disorders

Somatostatin is a tetradeca peptide hormone produced by many different endocrine cells throughout the body. It is also present in both the central and peripheral nervous system. The peptide has many different moods of action including inhibition of hormone secretion and influence on gastrointestinal motility. Somatostatin was identified in 1973 and about 10 years later the first long-acting analogue, octreotide, became available for use in clinical trials. Somatostatin analogues have been used to treat patients with neuroendocrine tumors, such as carcinoid tumors and endocrine pancreatic tumors, with symptoms due to excessive hormone production. It also has a well-documented effect on hormone levels and symptoms in acromegalic patients, while the use in diabetes mellitus is less well established. Several new analogues have been developed and tested for clinical use and lately non-peptide analogues have been produced. These new somatostatin receptor subtype specific analogues will soon be tested in clinical trials. In this review article the development of new analogues and new preparations of old analogues and their use in the clinic is discussed.

Structure-Based Design of a Novel Series of Nonpeptide Ligands That Bind to the pp60src SH2 Domain

The SH2 domain of pp60c-src (Src), a nonreceptor tyrosine kinase, facilitates signal transduction in a number of cell types through binding to cognate phosphorylated protein sequences. Phosphotyrosine-containing peptides have been shown to bind to the Src SH2 domain with micromolar affinity. Guided by the X-ray crystal structure of a phosphorylated peptide bound to the Src SH2 domain, we have designed a de novo series of small molecule ligands that bind with affinity comparable to the parent phosphopeptide. An X-ray crystal structure of the Src SH2 domain bound with a nonpeptide analog from this series verifies interactions targeted in the molecular design. However, a unique mode of binding has been revealed for the P-site phenyl phosphate group of the nonpeptide that differs from that observed for the phosphotyrosine side chain in peptide ligands bound to the Src SH2 domain. This novel binding mode is being used in guiding future design efforts.

Peptidic and Non-peptidic Neurotensin Analogs

The structure-activity relationship studies of neurotensin and its analogs have revealed that i) the C-terminal hexapeptide NT (8-13) (H-ArgB­ Arg9-Pro1o_Tyr1LIIe12-Leu13.QH) is equipotent to or more potent than the native NT(1-13) and retains all the intrinsic biological properties of neurotensin, ii) the N-terminal peptide fragment up to NT(1-7) does not significantly contribute to the binding potency, iii) Pro1o that can form a reverse-turn conformation is required, iv) an aromatic amino acid is important at Tyr11 position, v) the lipophilic side chain of Leu 13 is also crucial to the biological activities, and, vi) L-form amino acids are essential at critical positions for the binding potency. In addition to many peptidic neurotensin analogs, highly potent, orally active, and selective non-peptidic analogs as antagonists of NT receptors, which have long lasting effects and the ability to cross the blood­ brain barrier (e. g. SR 142948 A and SR 48692), have been discovered.

Potential applications of GH secretagogs in the evaluation and treatment of the age-related decline in growth hormone secretion.

The two classes of GH secretagogs--GH-releasing hormone (GHRH) and the GH-releasing peptides and their analogs (GHRP's)--retain their ability to endogenous GH secretion in healthy and frail elderly subjects. They have very limited utility in assessment of the state of the GH/IGF-I axis except to confirm an intact pituitary, but they are attractive potential alternatives to GH as therapeutic agents. There is wide interest in the possibility that elevating GH and IGF-I might increase muscle mass, physical strength and performance, and possible sleep and cognition in aging. The GH secretagogs, like GH, can produce a sustained stimulation of this axis; in contrast to GH, they preserve feedback regulation at the pituitary level and stimulate a near-physiologic pulsatile pattern of GH release. GHRP's and their nonpeptide analogs are also active when given orally, a significant practical advantage. Short-term treatment studies have shown that GHRH and the GHRP's can enhance GH secretion and elevate IGF-I and IGFBP-3 levels; that GHRH may promote sleep; and that these agents are generally well tolerated. Longer-term studies assessing effects upon body composition and physical and psychological function are underway.

Design, synthesis and pharmacological evaluation of active pyrrole based, nonpeptidic analogues of neurotensin(8-13)

We report the design, synthesis and pharmacological analysis of pyrrole based, nonpeptidic analogues of neurotensin(8-13) for testing further our reported multiple template approach to developing nonpeptidic mimetics of neuropeptides and for developing nonpeptidic mimetics of neurotensin(8-13) as potential drug candidates for the treatments of neuropsychiatric diseases such as schizophrenia and Parkinson’s disease. Three newly synthesized pyrrole analogues (mimics-4, -5 and -6) designed by the multiple template approach have been found to be more active in binding to the neurotensin receptor than the previously reported indole based mimics-1 and -2, which are a partial nonpeptidic antagonist and an agonist of neurotensin(8-13), respectively. The results support the theory of the multiple template approach and provide insights into the rational design of more potent neurotensin mimetics and into the library design for rational screening for effective nonpeptide compounds by combinatorial chemistry.

Sex steroid modulation of AT2 receptors in human myometrium.

In contrast to the abundant expression of the AT2 subtype of angiotensin II (AII) receptors during fetal development, AT2 receptor in adult life is expressed in few tissues. We now report studies on the presence and hormonal regulation of AT2 receptor in human pregnant and nonpregnant myometrium obtained from a large study population (n = 50). AT2 receptor subtypes have been characterized using self- and cross-competition curves among [125I]CGP42112A (a selective AT2 ligand), [125I](Sar1,Ile8)AII (a unselective antagonist), the corresponding unlabeled ligands, and several peptidic and nonpeptidic analogs with different affinities for the AT1 and AT2 receptor subtypes. We found that the human nonpregnant uterus expresses almost exclusively the AT2 subtype, and that [125I]CGP42112A is a selective probe to study human AT2 receptor. By using [125I]CGP42112A, we demonstrated that the density of AT2 receptor in human myometrium is dramatically affected by the hormonal milieu. Indeed, in the estrogen-dominant uterus of normal cycling women in the proliferative phase and that of perimenopausal women with anovulatory cycles, the density of binding sites was very high [1565 +/- 246 fmol/mg protein (n = 11) and 2176 +/- 429 (n = 7), respectively]. The concomitant presence of progestogens blunted the estrogen effect [term pregnancy, 61 +/- 12 fmol/mg protein (n = 5); secretive phase of the cycle, 453 +/- 154 (n = 10); combined oral contraceptive, 243 +/- 74 fmol/mg protein (n = 6)]. Very low concentrations of binding sites are also present in the sex steroid-deprived uterus of postmenopausal women (100 +/- 12 fmol/mg protein; n = 8) and the uterus of fertile women chronically treated with GnRH agonists (199 +/- 100 fmol/mg protein; n = 3). Hence, these data confirm the presence of AT2 receptors in human uterus and indicate their regulation by sex steroids.

The Active Site Specificity of the Yersinia Protein-tyrosine Phosphatase

Yersinia protein-tyrosine phosphatase substrates have been synthesized employing an expedient methodology that incorporates phosphorylated non-amino acid residues into an active site-directed peptide. While the peptidic portion of these compounds serves an enzyme targeting role, the nonpeptidic component provides a critical assessment of the range of functionality that can be accommodated within the active site region. We have found that the Yersinia phosphatase hydrolyzes both L- and D-stereoisomers of phosphotyrosine in active site-directed peptides, with the former serving as a 10-fold more efficient substrate than the latter. In addition, this enzyme catalyzes the hydrolysis of a variety of aromatic and aliphatic phosphates. Indeed, a peptide bearing the achiral phosphotyrosine analog, phosphotyramine, is not only the most efficient substrate described in this study, it is also one of the most efficient substrates ever reported for the Yersinia phosphatase. Straight chain peptide-bound aliphatic phosphates of the general structure, (Glu)4-NH-(CH2)n-OPO3(2-) (n = 2-8), are also hydrolyzed, where the most efficient substrate contains seven methylene groups. Finally, a comparison of the substrate efficacy of the peptide-bound species with that of the corresponding non-peptidic analogs, reveals that the peptide component enhances kcat/Km by up to nearly 3 orders of magnitude.

Modelling constrained calcitonin gene-related peptide analogues.

The calcitonin gene-related peptide (CGRP) is a 37-residue peptide with pronounced physiological activities, making it an interesting lead for drug development. In our previous work, we designed constrained analogues of the peptide. The biological activities of these proved that the disulfide bridges created favoured the active conformations of the peptide. Here we report the molecular modelling of the conformation of domains involved in receptor activation. Our approach was to create sets of possible conformations for three differently constrained bioactive regions by a random search of conformational space and molecular modelling. The conformations were compared, and those allowed for all derivatives were considered relevant for receptor activation. This approach resulted in one favoured group of conformations characterized by an inverse gamma-turn followed by a gamma-turn, defining the conformation of the tripeptide region with a similar sequence in all active analogues. The models may also be used for the design of peptide and non-peptide analogues of CGRP.

Low molecular weight neurokinin NK 2 antagonists

A deletion - optimization strategy based on an initial heptapeptide lead structure 1 led to potent and selective neurokinin NK 2 antagonists 5 (pK B=9.3) and 9 (pK B=7.9) of substantially reduced molecular size. Tetrapeptide 5 (0.1 μmol/Kg i.v.) potently inhibits NK 2 agonist-induced bronchoconstriction in guinea-pigs. Whilst less potent than 5 in vivo, the dipeptoid 9 (5 μmol/Kg i.v.) had a significantly longer biological half-life (> 2 h), and provides a potential lead towards non-peptide analogues.

Refinement of a molecular model of angiotensin II (AII) employed in the discovery of potent nonpeptide antagonists

A novel conformational model of AII, Model II, had been employed previously in the design of potent benzylimidazole AII antagonists ( J. Med. Chem. 1991, 34, 1514–1517). This paper considers this model in relation to the recently described potent AII analogs [hCys 3,5]-AII, and [Sar 1,hCys 3,5,IIe 8]-AII. Conformational analysis of Ac-S,S-cyclo-(hCys-Ala-hCys)-NH 2 suggests a family of modified conformations for AII, Model III, which retains the topological arrangement of functional groups in Model II that had been employed in nonpeptide analog design.

Molecular determinants of recognition and activation at the ?-opioid receptor by met-enkephalin-like peptides

In this study, the techniques of computational chemistry were used to probe the origin of the differing pharmacological profiles found as a result of two simple modifications of the enkephalin analog Tyr–DAla–Gly–Phe–MetNH2: the presence or absence of N-methylation of Phe4 and of the Met5 residue. Although all four analogs have high μ-receptor affinity, their analgesic activity varies by a factor of 3000. Thus, they should share common determinants of μ-receptor recognition while differing in the ability to activate the receptor. To identify and characterize these determinants, a two-step procedure was used. In the first step, the energy conformational profile of each peptide was obtained. The strategy used involved the iterative calculation of molecular dynamics trajectories at high and low temperatures, coupled to energy minimizations, allowing a through sampling of conformational space. In the second step, low-energy conformers of the four peptides were examined for the extent to which they fulfilled the requirements for μ-receptor recognition recently developed for nonpeptide analogs. In these studies, the amine nitrogen, a second proton-accepting moiety, and an aromatic ring in a specific geometric arrangement were proposed as the minimum components of a μ-pharmacophore for recognition. For all four analogs, a unique low-energy conformer was found that contained these three recognition moieties in a geometric arrangement to interact with the same target binding site residues as in the nonpeptide analogs. These results are consistent with the finding of high affinity for all four peptides and provide common determinants of recognition of the μ-receptor by peptides and nonpeptides. When the four peptides were overlapped so that they could each interact with these three common recognition sites, the Phe4 aromatic side chain was found to be a possible modulator of activation. For the parent pentapeptide, Tyr–DAla–Gly–Phe–Met, with the lowest activity, there was poor overlap of the Phe4 aromatic ring with the same ring in the other three analogs. These results implicate the Phe4 ring in peptide activation of the μ-receptor. © 1993 John Wiley & Sons, Inc.

Platelets and cancer metastasis: more than an epiphenomenon.

In this review we have presented overwhelming evidence that platelets play an important enhancing role in tumor cell-vasculature interactions and cancer metastasis. Tumor cells employ a host of mechanisms to induce platelet aggregation; vice versa, platelets make use of a spectrum of mechanisms to modulate tumor cell-EC and tumor cell-ECM interactions. Various COX and LOX metabolites are closely involved in this bidirectional modulation, thus providing us with a very promising therapeutic avenue by modifying the actions of these metabolites. On the other hand, adhesion molecules are mediators of tumor cell-platelet-EC interactions; therefore interference with these adhesion molecules and various adhesive processes either by soluble peptides, nonpeptidic analogues, or specific antibodies holds great investigational and clinical appeal.

Construction and expression of a monomeric c-Jun protein that binds and activates transcription of AP-1-responsive genes.

c-Jun is a typical member of the bZIP (basic zipper) family of dimeric transcriptional activators. These proteins contain a basic region responsible for DNA sequence recognition and a leucine zipper that mediates dimerization. bZIP proteins regulate a large number of important physiological functions and, therefore, present an interesting target for molecular interference and mimicry. As a step toward the development of peptide and nonpeptide analogs of such proteins, we constructed a derivative of c-Jun that binds DNA as a monomer. This construction was done by connecting a second basic region to the natural basic region of c-Jun by means of a short peptide loop. Although the polypeptide backbone of the second basic region has an inverted polarity relative to that of the natural basic region, the monomeric c-Jun protein binds DNA with reasonably high affinity and indistinguishable specificity from the wild-type, dimeric c-Jun protein. Furthermore, the monomeric c-Jun protein can activate transcription in vivo. These findings indicate that the polypeptide backbone of the basic region contributes little to sequence recognition and that the leucine zipper is not directly involved in transcriptional activation.