Discovery of a non-peptide small molecule that selectively mimics the biological actions of calcitonin

Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8-32), whereas SUN B8155 did not displace the specific binding of [(125)I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.     

Designed molecules that fold to mimic protein secondary structures

Molecules that fold to mimic protein secondary structures have emerged as important targets of bioorganic chemistry. Recently, a variety of compounds that mimic helices, turns, and sheets have been developed, with notable advances in the design of beta-peptides that mimic each of these structures. These compounds hold promise as a step toward synthetic molecules with protein-like properties and as drugs that block protein-protein interactions.     

Discovery of a series of small molecules as potent histone deacetylase inhibitors

Abstract

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC₅₀ of 0.14?μM and tumor cell growth inhibition IC₅₀ of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.     

Discovery of novel sulfonated small molecules that inhibit vascular tube formation

Tumor-associated angiogenesis is a complex process that involves the interplay among several molecular players such as cell-surface heparan sulfate proteoglycans, vascular endothelial growth factors and their cognate receptors. PI-88, a highly sulfonated oligosaccharide, has been shown to have potent anti-angiogenic activity and is currently in clinical trials. However, one of the major drawbacks of large oligosaccharides such as PI-88 is that their synthesis often requires numerous complex synthetic steps. In this study, several novel polysulfonated small molecule carbohydrate mimetics, which can easily be synthesized in fewer steps, are identified as promising inhibitors of angiogenesis in an in vitro tube formation assay.     

Hemodialysis removes uremic toxins that alter the biological actions of endothelial cells

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure.     

Ghrelin has novel vascular actions that mimic PI 3-kinase-dependent actions of insulin to stimulate production of NO from endothelial cells

Ghrelin is an orexigenic peptide hormone secreted by the stomach. In patients with metabolic syndrome and low ghrelin levels, intra-arterial ghrelin administration acutely improves their endothelial dysfunction. Therefore, we hypothesized that ghrelin activates endothelial nitric oxide synthase (eNOS) in vascular endothelium, resulting in increased production of nitric oxide (NO) using signaling pathways shared in common with the insulin receptor. Similar to insulin, ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells (BAEC) in primary culture (assessed using NO-specific fluorescent dye 4,5-diaminofluorescein) in a time- and dose-dependent manner. Production of NO in response to ghrelin (100 nM, 10 min) in human aortic endothelial cells was blocked by pretreatment of cells with NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor], or (D-Lys3)-GHRP-6 (selective antagonist of ghrelin receptor GHSR-1a), as well as by knockdown of GHSR-1a using small-interfering (si) RNA (but not by mitogen/extracellular signal-regulated kinase inhibitor PD-98059). Moreover, ghrelin stimulated increased phosphorylation of Akt (Ser473) and eNOS (Akt phosphorylation site Ser1179) that was inhibitable by knockdown of GHSR-1a using siRNA or by pretreatment of cells with wortmannin but not with PD-98059. Ghrelin also stimulated phosphorylation of mitogen-activated protein (MAP) kinase in BAEC. However, unlike insulin, ghrelin did not stimulate MAP kinase-dependent secretion of the vasoconstrictor endothelin-1 from BAEC. We conclude that ghrelin has novel vascular actions to acutely stimulate production of NO in endothelium using a signaling pathway that involves GHSR-1a, PI 3-kinase, Akt, and eNOS. Our findings may be relevant to developing novel therapeutic strategies to treat diabetes and related diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance.     

The biological potency of a series of analogues of human calcitonin correlates with their interactions with phospholipids

The conformational and lipid binding properties of several calcitonin analogs were compared. These analogs were designed to have the central amphipathic helical region of salmon calcitonin and N- and C-terminal segments similar to human calcitonin. The various analogs differed from one another either by removal of Leu19 from this hybrid analog, replacement of Leu19 with Gly19 or having a carboxyl terminus more closely related to salmon calcitonin. It had been found that replacement of Leu19 with Gly19 caused a marked reduction in the hypocalemic activity of the analog. The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide.     

Discovery of small molecules that inhibit melanogenesis via regulation of tyrosinase expression

5,6,7,8-Tetrahydro-4H-cyclohepta[d]isoxazole derivatives were synthesized and evaluated as a novel class of inhibitors for α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis in a mouse melanoma B16F10 cell line. Compound 8e (IC₅₀ = 0.67 μM), 8h (IC₅₀ = 1.01 μM) and 9b (IC₅₀ = 0.99 μM) exhibited a potent inhibitory activity approximately 85- to 126-fold greater than kojic acid, a well-known potent inhibitor. A biochemical study indicates that the activity of this series should be displayed via down-regulation of the expression of tyrosinase.     

Discovery of new small molecules that influence neuroblast cell migration from the subventricular zone

Using SVZ (subventricular zone) tissue explants from one-day-old mice, we investigated the activity of new amino aromatic disulfide analogues and polyazamacrocycles on the migration of SVZ cells (neuroblasts). We found that among the tested analogues, non-peptidic disulfide derivative 8 significantly decreases the migration of neuroblasts from SVZ cells, and antagonized the stimulating activity of disulfide cyclic peptide 1. Discovery of compounds 1 and 8 constitutes new chemical tools which could be used to understand the mechanism of neuroblast migration during neurogenesis and eventually to identify specific genes involved in the neurogenesis.     

Biological actions of human and rat calcitonin and calcitonin gene-related peptide

We assessed the central and peripheral biological actions of human and rat calcitonin and calcitonin gene-related peptide (CGRP). After intravenous administration, human and rat calcitonin, but neither human nor rat CGRP significantly decreased plasma calcium and phosphorus concentrations in awake, freely moving rats. After intracerebroventricular as well as after intravenous administration, human and rat calcitonin and human and rat CGRP significantly inhibited gastric acid secretion in conscious rats. Intracerebroventricular administration of rat calcitonin did not alter plasma calcium and phosphorus concentrations. Linear, partially protected CGRP and calcitonin did not exhibit any biological effects. These studies indicate that calcitonin, but not CGRP, affects calcium and phosphorus homeostasis while both peptides decrease gastric acid secretion similarly. Furthermore, these studies support the hypothesis that the calcium and phosphorus lowering effects of calcitonin are peripheral while the gastric inhibiting actions of the calcitonin and CGRP are mediated by the central nervous system.     

Juvadecene: Discovery of a juvenile hormone mimic in the plant,Macropiper excelsum

A compound with significant insect juvenile hormone activity was isolated from the plant, Macropiper excelsum. The chemical structure was determined by spectral methods to be 1-(3,4-methylenedioxyphenyl)-trans-3-decene(l), and confirmed by synthesis. The hormonally active substance applied topically to last (fifth)-instar nymphs of the milkweed bug (Oncopeltus fasciatus) induced a supernumerary metamorphosis at 30 μg. Higher doses were toxic.     

The protein-tethered lipid bilayer: a novel mimic of the biological membrane

A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (CcO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.     

Discovery of small molecules that enhance astrocyte differentiation in rat fetal neural stem cells

1,3,4-Oxadiazole derivatives were found to enhance astrocyte differentiation in rat fetal neural stem cells (NSCs). Differentiation activity was assessed by immunocytochemistry and analysis of mRNA expression of astrocyte markers, GFAP and S100. Compounds 7 and 8 showed approximately a two-fold increase in astrocyte differentiation without engagement of neuronal differentiation and detectable cytotoxicity.     

Discovery of a novel series of CXCR3 antagonists

The discovery of a novel series of CXCR3 antagonists is described. Starting from an HTS positive, iterative optimization gave potent compounds (IC₅₀ 15 nM in a chemotaxis assay). The strategy employed to improve the metabolic stability of these derivatives is described.     

Discovery of a novel series of Notch-sparing gamma-secretase inhibitors

Using a cell-based assay, we have identified a new series of Notch-sparing γ-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved γ-secretase inhibitory potency and Notch-sparing selectivity.     

Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells

CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-gamma production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.     

Some biological characteristics of lipid conjugates of protein antigen that selectively induce delayed-type hypersensitivity in mice

Covalent coupling of lipid to protein antigen, BSA, modifies the immunogenicity leading to selective induction of delayed type hypersensitivity in mice with no or very little concomitant antibody production. The mode of linkage of lipid to protein, however, controls its tissue distribution and retention in the body and cell uptake in vitro. Whereas D-BSA accumulates in the draining lymph node after foot pad inoculation, DA-BSA stays at the site. Further, DA-BSA is eliminated much more slowly and D-BSA more quickly from the body than the native antigen. Although both lipid-conjugates are taken up by lymphoid cells in vitro more than the native antigen, DA-BSA binds significantly more than D-BSA. On the basis of in vitro blastogenic response and enumeration of antigen sensitive cells and in vivo tests of delayed type hypersensitivity, DA-BSA appears superior to D-BSA but neither was as potent as BSA in CFA.     

Discovery of a novel series of selective HCN1 blockers

The discovery of a series of novel, potent, and selective blockers of the cyclic nucleotide-modulated channel HCN1 is disclosed. Here we report an SAR study around a series of selective blockers of the HCN1 channel. Utilization of a high-throughput VIPR assay led to the identification of a novel series of 2,2-disubstituted indane derivatives, which had moderate selectivity and potency at HCN1. Optimization of this hit led to the identification of the potent, 1,1-disubstituted cyclohexane HCN1 blocker, 2-ethoxy-N-((1-(4-isopropylpiperazin-1-yl)cyclohexyl)methyl)benzamide. The work leading to the discovery of this compound is described herein.     

Discovery of a highly potent series of TLR7 agonists

The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.