Interactions between integrase inhibitors and human arginase 1.

The neuro-pathogenic mechanism(s) underlying HIV-associated neurocognitive disorders are mostly unknown. HIV-infected macrophages and microglial cells play a crucial role and the metabolic fate of l-arginine may be highly relevant to microglia activation. In this context, arginase (ARG), which uses l-arginine as substrate, can be on the same time a target and source of oxidative stress and inflammation. In this study, we investigated whether integrase strand transfer inhibitors share with the other antiretroviral drugs the ability to inhibit ARG activity. We used the previously validated cell model, namely the human microglia cell line, as well as the computational chemistry approach. Furthermore, here we characterized the activity of purified human ARG in a cell-free in vitro system, and investigated the effects of integrase strand transfer inhibitors in this newly validated model. Overall evidence shows that Dolutegravir, Raltegravir and Elvitegravir inhibit ARG activity.

Antiretrovirals inhibit arginase in human microglia.

Preliminary evidence in an animal model, that is, primary cultures of rat microglia cells, suggested that some antiretroviral drugs (ARVs), namely darunavir, atazanavir, efavirenz, and nevirapine, increase NO production through a mechanism involving the inhibition of arginase (ARG) activity. This study was conceived to investigate the effects of ARVs on ARG activity in a human experimental model. We compared CHME-5 human microglial immortalized cells under basal conditions with cells exposed to either IL-4, a mix of inflammatory cytokines, or both stimuli given together. We also tested the effects of ARVs on CHME-5 cell lysates after exposure to the above stimuli. Moreover, the interaction between the ARVs and ARG was investigated via computational chemistry. We found that ARVs consistently inhibit ARG activity both in intact and lysed cells. In docking studies, darunavir and atazanavir showed similar scores compared with both l-arginine and the ARG antagonist nor-NOHA. Efavirenz and nevirapine, which are less potent in inhibiting ARG in the biochemical assay, also had lower scores. In conclusion, the present findings in a human model support the notion that ARG pathway can present a new, additional molecular target for different ARVs in HIV treatments. We found that antiretroviral drugs (ARVs) consistently inhibit arginase (ARG)-I activity both in intact and lysed cells. In docking studies, darunavir (DRV) and atazanavir (ATV) showed similar scores compared to both l-arginine and the ARG antagonist, Nω-hydroxy-nor-arginine (nor-NOHA). Efavirenz (EFV) and nevirapine (NVP), which are less potent in inhibiting ARG in the biochemical assay, also had lower scores. In conclusion, the present findings in a human model support the notion that ARG pathway can be envisioned as an additional and new molecular target of different ARVs in HIV treatments.

Recent Advances on Type-2 Cannabinoid (CB2) Receptor Agonists and their Therapeutic Potential.

In the last decade, selective modulators of type-2 cannabinoid receptor (CB2) have become a major focus to target endocannabinoid signaling in humans. Indeed, heterogeneously expressed within our body, CB2 actively regulates several physio-pathological processes, thus representing a promising target for developing specific and safe therapeutic drugs. If CB2 modulation has been extensively studied since the very beginning for the treatment of pain and inflammation, the more recent involvement of this receptor in other pathological conditions has further strengthened the pursuit of novel CB2 agonists in the last five years. Against this background, here we discuss the most recent evidence of the protective effects of CB2 against pathological conditions, emphasizing central nervous system disorders, bone and synovial diseases, and cancer. We also summarize the most recent advances in the development of CB2 agonists, focusing on the correlation between different chemical classes and diverse therapeutic applications. Data mining includes a review of the CB2 ligands disclosed in patents also released in the last five years. Finally, we discuss how the recent elucidation of CB2 tertiary structure has provided new details for the rational design of novel and more selective CB2 agonists, thus supporting innovative strategies to develop effective therapeutics. Our overview of the current knowledge on CB2 agonists provides pivotal information on the structure and function of different classes of molecules and opens possible avenues for future research.

Exploring the 1,3-benzoxazine chemotype for cannabinoid receptor 2 as a promising anti-cancer therapeutic.

The discovery of selective agonists of cannabinoid receptor 2 (CB2) is strongly pursued to successfully tuning endocannabinoid signaling for therapeutic purposes. However, the design of selective CB2 agonists is still challenging because of the high homology with the cannabinoid receptor 1 (CB1) and for the yet unclear molecular basis of the agonist/antagonist switch. Here, the 1,3-benzoxazine scaffold is presented as a versatile chemotype for the design of CB2 agonists from which 25 derivatives were synthesized. Among these, compound 7b5 (CB2 EC50 = 110 nM, CB1 EC50 > 10 µM) demonstrated to impair proliferation of triple negative breast cancer BT549 cells and to attenuate the release of pro-inflammatory cytokines in a CB2-dependent manner. Furthermore, 7b5 abrogated the activation of extracellular signal-regulated kinase (ERK) 1/2, a key pro-inflammatory and oncogenic enzyme. Finally, molecular dynamics studies suggested a new rationale for the in vitro measured selectivity and for the observed agonist behavior.

In silico scaffold evaluation and solid phase approach to identify new gelatinase inhibitors.

Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents.

Stability Evaluation and Degradation Studies of DAC® Hyaluronic-Polylactide Based Hydrogel by DOSY NMR Spectroscopy.

The stability and the degradation of polymers in physiological conditions are very important issues in biomedical applications. The copolymer of hyaluronic acid and poly-D,L-lactic acid (made available in a product called DAC®) produces a hydrogel which retains the hydrophobic character of the poly-D,L-lactide sidechains and the hydrophilic character of a hyaluronic acid backbone. This hydrogel is a suitable device for the coating of orthopedic implants with structured surfaces. In fact, this gel creates a temporary barrier to bacterial adhesion by inhibiting colonization, thus preventing the formation of the biofilm and the onset of an infection. Reabsorbed in about 72 h after the implant, this hydrogel does not hinder bone growth processes. In the need to assess stability and degradation of both the hyaluronan backbone and of the polylactic chains along time and temperature, we identified NMR spectroscopy as a privileged technique for the characterization of the released species, and we applied diffusion-ordered NMR spectroscopy (DOSY-NMR) for the investigation of molecular weight dispersion. Our diffusion studies of DAC® in physiological conditions provided a full understanding of the product degradation by overcoming the limitations observed in applying classical chromatography approaches by gel permeation UV.

Structural bases for substrate and inhibitor recognition by matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which are involved in the proteolytic processing of several components of the extracellular matrix. As a consequence, MMPs are implicated in several physiological and pathological processes, like skeletal growth and remodelling, wound healing, cancer, arthritis, and multiple sclerosis, raising a very widespread interest toward this class of enzymes as potential therapeutic targets. Here, structure-function relationships are discussed to highlight the role of different MMP domains on substrate/inhibitor recognition and processing and to attempt the formulation of advanced guidelines, based on natural substrates, for the design of inhibitors more efficient in vivo.

Nitazoxanide inhibits paramyxovirus replication by targeting the Fusion protein folding: role of glycoprotein-specific thiol oxidoreductase ERp57.

Paramyxoviridae, a large family of enveloped viruses harboring a nonsegmented negative-sense RNA genome, include important human pathogens as measles, mumps, respiratory syncytial virus (RSV), parainfluenza viruses, and henipaviruses, which cause some of the deadliest emerging zoonoses. There is no effective antiviral chemotherapy for most of these pathogens. Paramyxoviruses evolved a sophisticated membrane-fusion machine consisting of receptor-binding proteins and the fusion F-protein, critical for virus infectivity. Herein we identify the antiprotozoal/antimicrobial nitazoxanide as a potential anti-paramyxovirus drug targeting the F-protein. We show that nitazoxanide and its circulating-metabolite tizoxanide act at post-entry level by provoking Sendai virus and RSV F-protein aggregate formation, halting F-trafficking to the host plasma membrane. F-protein folding depends on ER-resident glycoprotein-specific thiol-oxidoreductase ERp57 for correct disulfide-bond architecture. We found that tizoxanide behaves as an ERp57 non-competitive inhibitor; the putative drug binding-site was located at the ERp57-b/b' non-catalytic domains interface. ERp57-silencing mimicked thiazolide-induced F-protein alterations, suggesting an important role of this foldase in thiazolides anti-paramyxovirus activity. Nitazoxanide is used in the clinic as a safe and effective antiprotozoal/antimicrobial drug; its antiviral activity was shown in patients infected with hepatitis-C virus, rotavirus and influenza viruses. Our results now suggest that nitazoxanide may be effective also against paramyxovirus infection.

Design, Synthesis, and Biological Evaluation of Tetrahydro-ß-carboline Derivatives as Selective Sub-Nanomolar Gelatinase Inhibitors.

Targeting matrix metalloproteinases (MMPs) is a pursued strategy for treating several pathological conditions, such as multiple sclerosis and cancer. Herein, a series of novel tetrahydro-ß-carboline derivatives with outstanding inhibitory activity toward MMPs are present. In particular, compounds 9 f, 9 g, 9 h and 9 i show sub-nanomolar IC50 values. Interestingly, compounds 9 g and 9 i also provide remarkable selectivity toward gelatinases; IC50 =0.15 nm for both toward MMP-2 and IC50 =0.63 and 0.58 nm, respectively, toward MMP-9. Molecular docking simulations, performed by employing quantum mechanics based partial charges, shed light on the rationale behind binding involving specific interactions with key residues of S1' and S3' domains. Taken together, these studies indicate that tetrahydro-ß-carboline represents a promising scaffold for the design of novel inhibitors able to target MMPs and selectively bias gelatinases, over the desirable range of the pharmacokinetics spectrum.

2-Arylpropionic CXC chemokine receptor 1 (CXCR1) ligands as novel noncompetitive CXCL8 inhibitors.

The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We report here molecular modeling studies showing a putative interaction site of 1 in the TM region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimization of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia reperfusion injury.

GPR55: Current knowledge and future perspectives of a purported "Type-3" cannabinoid receptor.

In the last decade, accumulated evidence highlighted that GPR55 might be activated by several classical cannabinoid ligands, making this orphan receptor the main candidate to be considered as the "third" cannabinoid receptor. The investigation of its pharmacology has often provided divergent and more intricate results that have complicated the understanding of the physiological role of GPR55. Nevertheless, the patent analysis regarding GPR55 outlines the fair interest of big pharmaceutical companies, especially in the first years of this decade. This investigation provides a brief overview of the current "state of the art" of our knowledge of GPR55, giving particular emphasis to its functional selectivity. This property could account for controversial roles of GPR55, whose pharmacology and downstream signaling is known to vary significantly both in ligand- and system-dependent manners. In addition, we gain insights into the challenging aspect of finding out novel GPR55 modulators, by analyzing conserved structural and functional motifs that, together with future studies, could help to elucidate its mechanism of action and to design more selective and potent small-molecules directed towards GPR55. Preliminary data highlight remarkable differences, but also intriguing commonalities, between GPR55 and other members of class A G protein-coupled receptors. It is anticipated that, in the next future, novel lead candidates targeting GPR55 could represent new tools to better understand GPR55-mediated human diseases and, hopefully, generate an innovative class of effective next-generation therapeutics.

Thiopyrophosphoantigens: solid-phase synthesis and in vitro characterization of a new class of Vgamma9 Vdelta2 T cells activators.

The Vgamma9 Vdelta2 T cells mediate rapid, innate-like immune responses to pathogens and are important in several key immunoregulatory pathways, including those involved in infections and tumor development. Vgamma9 Vdelta2 T cells respond to low molecular weight isoprenoid phosphoantigens; the prototypic stimulatory compound is isopentenylpyrophosphate (IPP), an alkylphosphate intermediate of mevalonate metabolism that elicits proliferative, cytotoxic, and cytokine secretion responses. We studied the replacement of the pyrophosphate moiety with the thiopyrophosphate bioisostere, synthesizing thioanalogues of IPP and 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP, the most potent natural antigen known to date). Once their in vitro efficacy and stability had been demonstrated, we synthesized a small library of compounds through the development of an innovative solid-phase strategy. Biological results confirmed thioHMBPP to be the best compound of this first series. Future aims are (i) the exploitation of the parallel solid-phase strategy to further explore structure-activity relationships of this new class of synthetic antigens and (ii) the determination of the PK/PD profile of thioHMBPP.