Functional characterization of the modified melanocortin peptides responsible for ligand selectivity at the human melanocortin receptors

The melanocortin system plays an important role in energy homeostasis as well as skin pigmentation, steroidogenesis and exocrine gland function. In this study, we examined eight Ac-His-Phe-Arg-Trp-NH(2) tetrapeptides that were modified at the Phe position and pharmacologically characterized their activities at the human MCR wild-types and their mutants. Our results indicate that at the hMC1R, all D stereochemical modified residues at the Phe position of peptides increase cAMP production in a dose-dependent manner. At the hMC3R, the DPhe peptide dose dependently increases cAMP production but all other three tetrapeptides were not. At the hMC4R, both the DPhe and DNal(1') peptides induce cAMP production. However, both DTyr and DNal(2') were not able to induce cAMP production. Further studies indicated that at the hMC1R M128L mutant receptor, the all D-configured tetrapeptides reduce their potencies as compared to that of hMC1R wild-type. However, at the hMC3R and hMC4R L165M and L133M mutant receptors, the DNal(2') and DTyr tetrapeptides possess agonist activity. These findings indicate that DPhe in tetrapeptide plays an important role in ligand selectivity and specific residue TM3 of the melanocortin receptors is crucial for ligand selectivity.     

Synthesis and evaluation of [(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]cyclohexanes and 4-[(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]piperidines as DPP-4 inhibitors

A series of 4-amino cyclohexanes and 4-substituted piperidines were prepared and evaluated for inhibition of DPP-4. Analog 20q displayed both good DPP-4 potency and selectivity against other proteases, while derivative 20k displayed long half life and modest oral bioavailability in rat. The most potent analog, 3-(5-aminocarbonylpyridyl piperidine 53j, displayed excellent DPP-4 activity with good selectivity versus other proline enzymes.     

Structure-activity relationship of linear peptide Bu-His6-DPhe7-Arg8-Trp9-Gly10-NH2 at the human melanocortin-1 and -4 receptors: DPhe7 and Trp9 substitution

A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.     

Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. Identification of 2-azabicyclo[4.1.0]heptan-3-imines

Further chemical modification of 2-iminopiperidines fused to cyclopropane rings was performed. Optically active isomers 2 and 13 were synthesized and their biological activity was evaluated. Compound 2 exhibited greater potency and more isoform selectivity than enantiomer 13 in the iNOS inhibition assay. One of the gem-chlorines on the fused cyclopropane moiety of 2 was eliminated to produce 3, which showed reduced potency for iNOS inhibition, as well as 4 with an increased potency. The isoform selectivity of 4 was also much higher than that of 3. This was also true for the corresponding methyl derivatives 6-9. The structure-activity relationship (SAR) study and computer aided docking study of the most optimized structure 4 with human iNOS will also be reported.     

Structure-activity relationship studies on 2-heteroaryl-4-arylimidazoles NPY5 receptor antagonists

A series of 2-heteroaryl-4-arylimidazoles with potent in vitro activity at the NPY5 receptor was developed. Introduction of electron-withdrawing groups on the 4-aryl ring led to a significant improvement of in vitro potency. Several analogues from this series had anorectic activity in rodent feeding models, but were also found to have undesired behavioral effects in spontaneous locomotor activity.     

Parallel synthesis and SAR study of novel oxa-steroids as potent and selective progesterone receptor antagonists

Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl and heterocycles, were evaluated for progesterone receptor (PR) and glucocorticoid receptor (GR) antagonist activities. SAR study demonstrated that the para-fluorinated substituents on the phenyl ring not only increased the potency for PR in a T47D cell functional assay, but also improved the selectivity over GR in an A549 cell functional assay. The para-fluorophenyl oxa-steroid 4l and the para-trifluoromethylphenyl oxa-steroid 4p were found to be remarkably more potent and more selective PR antagonists than mifepristone, with subnanomolar potency and about 140-fold selectivity over GR. Molecular modeling of the oxa-steroid bound to PR provided meaningful insight for the SAR study. oxa-Steroids 4a and 4b were found to be more efficacious than mifepristone in vivo in a rat uterine complement C3 assay via the oral route, although they were less than or equally potent to mifepristone in the T47D assay.     

Novel thiol-based TACE inhibitors: rational design, synthesis, and SAR of thiol-containing aryl sulfonamides

A series of potent thiol-containing aryl sulfonamide TACE inhibitors was designed and synthesized. The SAR and MMP selectivity of the series were investigated. In particular, compound 4b has shown excellent in vitro potency against the isolated TACE enzyme and good selectivity over MMP-2, -7, -8, -9, and -13. The X-ray structure of 4b bound to TACE was obtained.     

Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains. Demonstration of differential sensitivity to kinase inhibitors.

Epidermal growth factor receptor (EGFR), ErbB-2, and ErbB-4 are members of the type 1 receptor tyrosine kinase family. Overexpression of these receptors, especially ErbB-2 and EGFR, has been implicated in multiple forms of cancer. Inhibitors of EGFR tyrosine kinase activity are being evaluated clinically for cancer therapy. The potency and selectivity of these inhibitors may affect the efficacy and toxicity of therapy. Here we describe the expression, purification, and biochemical comparison of EGFR, ErbB-2, and ErbB-4 intracellular domains. Despite their high degree of sequence homology, the three enzymes have significantly different catalytic properties and substrate kinetics. For example, the catalytic activity of ErbB-2 is less stable than that of EGFR. ErbB-2 uses ATP-Mg as a substrate inefficiently compared with EGFR and ErbB-4. The three enzymes have very similar substrate preferences for three optimized peptide substrates, but differences in substrate synergies were observed. We have used the biochemical and kinetic parameters determined from these studies to develop an assay system that accurately measures inhibitor potency and selectivity between the type 1 receptor family. We report that the selectivity profile of molecules in the 4-anilinoquinazoline series can be modified through specific aniline substitutions. Moreover, these compounds have activity in whole cells that reflect the potency and selectivity of target inhibition determined with this assay system.     

CD22-antagonists with nanomolar potency: the synergistic effect of hydrophobic groups at C-2 and C-9 of sialic acid scaffold

In earlier studies, we identified the C-9 amido derivative 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) and the C-9 amino derivative 2 (9-(4'-hydroxy-4-biphenyl)methylamino-9-deoxy-Neu5Gcα2-6GalOMP) have the most promising affinity for mouse CD22 and human CD22, respectively. Replacing the subterminal galactose residue (2-6Gal-OMP) of 1 with benzyl (5) or biphenylmethyl (6) as aglycone led to even higher potency for mCD22. In this study, both compounds showed improved potency and selectivity for CD22 (IC(50) 70 nM) and 712-fold more selective for CD22 than for MAG. The corresponding derivatives of 2, compounds 8 and 9, showed comparable activity to 2 but lower potency and selectivity than 5 and 6. Although compounds 5-9 are simple and small molecular weight antagonists, they showed much high potency and selectivity than the corresponding compounds having α 2-6Gal linkage. Both biological and computational docking simulation studies suggest that the 2-6Gal-OMP residues of 1 and 2 are not critical for binding process and could be replaced with hydrophobic non-carbohydrate moieties. The data presented herein has significant implications for the design and discovery of next-generation CD22-antagonists.     

Analogs of MTII, lactam derivatives of alpha-melanotropin, modified at the N-terminus, and their selectivity at human melanocortin receptors 3, 4, and 5.

In search for selective agonists at human melanocortin-4 receptor, proline-substituted analogs of MTII, a potent nonselective agonist at melanocortin receptors, were prepared by solid-phase syntheses and evaluated for their ability to bind and activate human MC-3, MC-4, and MC-5 receptors. Replacement of Nle(4) with Pro resulted in [Pro(4)]MTII with affinity to and agonist potency at hMC-4R similar to MTII, but with about 400-fold lower potency at hMC-5R and about 20-fold lower potency at hMC-3R. The substantial increase in selectivity of [Pro(4)]MTII with respect to hMC-5R prompted us to investigate additional analogs of MTII with modified N-termini. The Ac-Nle(4) segment, not encompassed in the lactam ring, was substituted with flexible, hydrophobic, or hydrophilic substituents, and also, with residues resembling proline. The similar agonist potency of these peptides to that of MTII at hMC-4R but significantly lower activity of these compounds at hMC-5R demonstrated that the N-terminal fragment of MTII has virtually no effect on the binding affinity and activation at hMC-4R, but it is essential for full potency at hMC-5R.     

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe⁷ with d-Phe or d-naphthylalanine (d-Nal(2′)) in ACTH(1–24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe⁷ with d-Nal(2′) in ACTH(1–24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe⁷ with d-Phe⁷ in ACTH(1–17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in d-Phe ACTH binding affinity and potency. Our results suggest that Phe⁷ in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.     

Benzimidazole-based fXa inhibitors with improved thrombin and trypsin selectivity

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.     

Selectivity and competitive interactions between two benthic invertebrate grazers (Asellus aquaticus and Potamopyrgus antipodarum): an experimental study using 13C- and 15N-labelled diatoms

1. Tracer experiments with two diatoms labelled with ¹³C (Nitzschia palea) and ¹⁵N (Fragilaria crotonensis), were conducted to investigate feeding selectivity and interspecific competition between the grazers Asellus aquaticus (Isopoda, Crustacea) and Potamopyrgus antipodarum (Hydrobiidae, Gastropoda). Conventional methods, such as cell counts and estimated biovolume, were used first to detect feeding preferences within the different grazer treatments. 2. The results revealed a significant decline in algal biovolume in all grazer treatments and no indications of active selectivity were observed. In contrast to conventional methods, measurements based on isotope signatures showed strong differences in tracer uptake, thus indicating different degrees of assimilation and digestion by the two grazers. 3. The selectivity index Q, which provides information on the uptake ratio of ¹³C to ¹⁵N, showed a significant time effect for both grazer species and a significant difference between single‐ and mixed‐grazer treatments for P. antipodarum. Thus, this technique enabled the direct quantification of the uptake by grazers and, therefore, served as an ideal tool for the detection of passive selectivity. 4. Our results indicate a shift in feeding preferences related to between‐species competition and a potential divergence of trophic niches when species coexist.     

Discovery of benzothiazole-based adenosine A2B receptor antagonists with improved A2A selectivity

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A_2B antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A_2A and A₁ receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A_2A and A₁ receptors.     

Design of Gram-negative selective antimicrobial peptides

Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, signals bacterial invasion and triggers defensive host responses. However, excessive responses also lead to the serious pathophysiological consequence of septic shock. To develop Gram-negative selective compounds that can inhibit the effects of LPS-induced sepsis, we have designed constrained cyclic antimicrobial peptides based on a cystine-stabilized beta-stranded framework mimicking the putative LPS-binding sites of the LPS-binding protein family. Our prototype termed R4A, c(PACRCRAG-PARCRCAG), consists of an eight amino acid degenerated repeat constrained by a head-to-tail cyclic peptide backbone and two cross-bracing disulfides. NMR study of K4A, an R4A analogue with four Arg --> Lys replacements, confirmed the amphipathic design elements with four Lys on one face of the antiparallel beta-strand and two hydrophobic cystine pairs plus two Ala on the opposite face. K4A and R4A displayed moderate microbicidal potency and Gram-negative selectivity. However, R4A analogues with single or multiple replacements of Ala and Gly with Arg or bulky hydrophobic amino acids displayed increased potency and selectivity in both low- and high-salt conditions. Analogues R5L and R6Y containing additional cationic and bulky hydrophobic amino acids proved the best mimics of the amphipathic topology of the "active-site" beta-strands of LPS-binding proteins. They displayed potent activity against Gram-negative E. coli with a minimal inhibitory concentration of 20 nM and a >200-fold selectivity over Gram-positive S. aureus. Our results suggest that an LPS-targeted design may present an effective approach for preparing selective peptide antibiotics.     

Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors

A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.     

The identification of pyrimidine-diazabicyclo[3.3.0]octane derivatives as 5-HT2C receptor agonists

The 5-HT2C receptor has been implicated in the regulation of appetite. As such, small molecule agonists to this receptor may serve as novel therapies to combat obesity. We describe here the identification, synthesis, and SAR of a 5-HT2C agonist from a unique pyrimidine-diazabicyclo[3.3.0]octane series. This compound displayed good potency at the 5-HT2C receptor, modest selectivity relative to other 5-HT2 receptors, and was efficacious in an acute feeding study in rats.     

Pyridinesulfonylureas and pyridinesulfonamides as selective bombesin receptor subtype-3 (BRS-3) agonists

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor belonging to the subfamily of bombesin-like receptors. BRS-3 is implicated in the development of obesity and diabetes. We report here small-molecule agonists that are based on a 4-(alkylamino)pyridine-3-sulfonamide core. We describe the discovery of 2a, which has mid-nanomolar potency, selectivity for human BRS-3 versus the other bombesin-like receptors, and good bioavailability.     

Dual-site binding of bivalent 4-aminopyridine- and 4-aminoquinoline-based AChE inhibitors: contribution of the hydrophobic alkylene tether to monomer and dimer affinities

Three series of 4-aminopyridine-and 4-aminoquinoline based symmetrical bivalent acetylcholinesterase (AChE) inhibitors were prepared and compared to previously synthesized dimers of 9-amino-1,2,3,4-tetrahydroacridine (tacrine). In each case significant, tether length-dependent increases in AChE inhibition potency and selectivity (up to 3000-fold) were observed relative to the corresponding monomer, indicating dual-site binding of these inhibitors to AChE. Assay of the corresponding alkylated monomers revealed that the alkylene tether played at least two complementary roles in the dimer series. In addition to reducing the entropy loss that occurs on binding both monomeric units of the dimer, the alkylene tether can also significantly improve potency through hydrophobic effects.     

Structure-based design of new DHFR-based antibacterial agents: 7-aryl-2,4-diaminoquinazolines

Dihydrofolate reductase (DHFR) inhibitors such as trimethoprim (TMP) have long played a significant role in the treatment of bacterial infections. Not surprisingly, after decades of use there is now bacterial resistance to TMP and therefore a need to develop novel antibacterial agents with expanded spectrum including these resistant strains. In this study, we investigated the optimization of 2,4-diamnoquinazolines for antibacterial potency and selectivity. Using structure-based drug design, several 7-aryl-2,4-diaminoquinazolines were discovered that have excellent sub-100 picomolar potency against bacterial DHFR. These compounds have good antibacterial activity especially on gram-positive pathogens including TMP-resistant strains.