Discovery of the first non-ATP competitive IGF-1R kinase inhibitors: advantages in comparison with competitive inhibitors.

A new series of IGF-1R inhibitors related to hydantoins were identified from a lead originating from HTS. Their noncompetitive property as well as their slow binding characteristics provided a series of compounds with unique selectivity and excellent cellular activities.

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

Antiatherosclerotic effects of small-molecular-weight compounds enhancing endothelial nitric-oxide synthase (eNOS) expression and preventing eNOS uncoupling.

Many cardiovascular diseases are associated with reduced levels of bioactive nitric oxide (NO) and an uncoupling of oxygen reduction from NO synthesis in endothelial NO synthase (eNOS uncoupling). In human endothelial EA.hy 926 cells, two small-molecular-weight compounds with related structures, 4-fluoro-N-indan-2-yl-benzamide (CAS no. 291756-32-6; empirical formula C16H14FNO; AVE9488) and 2,2-difluoro-benzo[1,3]dioxole-5-carboxylic acid indan-2-ylamide (CAS no. 450348-85-3; empirical formula C17H13F2NO3; AVE3085), enhanced eNOS promoter activity in a concentration-dependent manner; with the responsible cis-element localized within the proximal 263 base pairs of the promoter region. RNA interference-mediated knockdown of the transcription factor Sp1 significantly reduced the basal activity of eNOS promoter, but it did not prevent the transcription activation by the compounds. Enhanced transcription of eNOS by AVE9488 in primary human umbilical vein endothelial cells was associated with increased levels of eNOS mRNA and protein expression, as well as increased bradykinin-stimulated NO production. In both wild-type C57BL/6J mice and apolipoprotein E-knockout (apoE-KO) mice, treatment with AVE9488 resulted in enhanced vascular eNOS expression. In apoE-KO mice, but not in eNOS-knockout mice, treatment with AVE9488 reduced cuff-induced neointima formation. A 12-week treatment with AVE9488 or AVE3085 reduced atherosclerotic plaque formation in apoE-KO mice, but not in apoE/eNOS-double knockout mice. Aortas from apoE-KO mice showed a significant generation of reactive oxygen species. This was partly prevented by nitric-oxide inhibitor N(omega)-nitro-l-arginine methyl ester, indicating eNOS uncoupling. Treatment of mice with AVE9488 enhanced vascular content of the essential eNOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin and reversed eNOS uncoupling. The combination of an up-regulated eNOS expression and a reversal of eNOS uncoupling is probably responsible for the observed vasoprotective properties of this new type of compounds.

Structural requirements for inhibition of the neuronal nitric oxide synthase (NOS-I): 3D-QSAR analysis of 4-oxo- and 4-amino-pteridine-based inhibitors.

The family of homodimeric nitric oxide synthases (NOS I-III) catalyzes the generation of the cellular messenger nitric oxide (NO) by oxidation of the substrate L-arginine. The rational design of specific NOS inhibitors is of therapeutic interest in regulating pathological NO levels associated with sepsis, inflammatory, and neurodegenerative diseases. The cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) maximally activates all NOSs and stabilizes enzyme quaternary structure by promoting and stabilizing dimerization. Here, we describe the synthesis and three-dimensional (3D) quantitative structure-activity relationship (QSAR) analysis of 65 novel 4-amino- and 4-oxo-pteridines (antipterins) as inhibitors targeting the H(4)Bip binding site of the neuronal NOS isoform (NOS-I). The experimental binding modes for two inhibitors complexed with the related endothelial NO synthase (NOS-III) reveal requirements of biological affinity and form the basis for ligand alignment. Different alignment rules were derived by building other compounds accordingly using manual superposition or a genetic algorithm for flexible superposition. Those alignments led to 3D-QSAR models (comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA)), which were validated using leave-one-out cross-validation, multiple analyses with two and five randomly chosen cross-validation groups, perturbation of biological activities by randomization or progressive scrambling, and external prediction. An iterative realignment procedure based on rigid field fit was used to improve the consistency of the resulting partial least squares models. This led to consistent and highly predictive 3D-QSAR models with good correlation coefficients for both CoMFA and CoMSIA, which correspond to experimentally determined NOS-II and -III H(4)Bip binding site topologies as well as to the NOS-I homology model binding site in terms of steric, electrostatic, and hydrophobic complementarity. These models provide clear guidelines and accurate activity predictions for novel NOS-I inhibitors.

Biochemistry and pharmacology of novel anthranilic acid derivatives activating heme-oxidized soluble guanylyl cyclase.

The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentration-dependent and quickly reversible fashion (EC50 = 0.5-10 microM), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of heme-oxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC50 1 to 10 microM); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.