Weijun Xu

Potent cationic inhibitors of West Nile virus NS2B/NS3 protease with serum stability, cell permeability and antiviral activity.

West Nile virus (WNV) has spread rapidly around the globe, efficiently crossing species from migrating birds into humans and other mammals. The viral protease NS2B-NS3 is important for WNV replication and recognizes dibasic substrate sequences common to other flaviviral proteases but different from most mammalian proteases. Potent inhibitors of WNV protease with antiviral activity have been elusive to date. We report the smallest and most potent inhibitors known for this enzyme, cationic tripeptides with nonpeptidic caps at the N-terminus and aldehyde at the C-terminus. One of these, compound 3 ( Ki = 9 nM) is stable in serum (>90% intact after 3 h, 37 degrees C), cell permeable, and shows antiviral activity (IC 50 1.6 microM) without cytotoxicity (IC 50 >400 microM), thereby validating the approach of inhibiting WNV protease to suppress WNV replication.

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells.

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals.

Orally absorbed cyclic peptides

Peptides and proteins are not orally bioavailable in mammals, although a few peptides are intestinally absorbed in small amounts. Polypeptides are generally too large and polar to passively diffuse through lipid membranes, while most known active transport mechanisms facilitate cell uptake of only very small peptides. Systematic evaluations of peptides with molecular weights above 500 Da are needed to identify parameters that influence oral bioavailability. Here we describe 125 cyclic peptides containing four to thirty-seven amino acids that are orally absorbed by mammals. Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable N- and C-termini and by shielding components from metabolic enzymes. Cyclization also folds peptides into bioactive conformations that determine exposure of polar atoms to solvation by water and lipids and therefore can influence oral bioavailability. Key chemical properties thought to influence oral absorption and bioavailability are analyzed, including molecular weight, octanol-water partitioning, hydrogen bond donors/acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally considered to be important for oral bioavailability of drug-like small molecules, although fewer hydrogen bond donors and reduced flexibility generally favored oral absorption.

Comparing sixteen scoring functions for predicting biological activities of ligands for protein targets

Accurately predicting relative binding affinities and biological potencies for ligands that interact with proteins remains a significant challenge for computational chemists. Most evaluations of docking and scoring algorithms have focused on enhancing ligand affinity for a protein by optimizing docking poses and enrichment factors during virtual screening. However, there is still relatively limited information on the accuracy of commercially available docking and scoring software programs for correctly predicting binding affinities and biological activities of structurally related inhibitors of different enzyme classes. Presented here is a comparative evaluation of eight molecular docking programs (Autodock Vina, Fitted, FlexX, Fred, Glide, GOLD, LibDock, MolDock) using sixteen docking and scoring functions to predict the rank-order activity of different ligand series for six pharmacologically important protein and enzyme targets (Factor Xa, Cdk2 kinase, Aurora A kinase, COX-2, pla2g2a, β Estrogen receptor). Use of Fitted gave an excellent correlation (Pearson 0.86, Spearman 0.91) between predicted and experimental binding only for Cdk2 kinase inhibitors. FlexX and GOLDScore produced good correlations (Pearson>0.6) for hydrophilic targets such as Factor Xa, Cdk2 kinase and Aurora A kinase. By contrast, pla2g2a and COX-2 emerged as difficult targets for scoring functions to predict ligand activities. Although possessing a high hydrophobicity in its binding site, β Estrogen receptor produced reasonable correlations using LibDock (Pearson 0.75, Spearman 0.68). These findings can assist medicinal chemists to better match scoring functions with ligand-target systems for hit-to-lead optimization using computer-aided drug design approaches.

Stabilizing short-lived Schiff base derivatives of 5-aminouracils that activate mucosal-associated invariant T cells

Mucosal-associated invariant T (MAIT) cells are activated by unstable antigens formed by reactions of 5-amino-6-D-ribitylaminouracil (a vitamin B2 biosynthetic intermediate) with glycolysis metabolites such as methylglyoxal. Here we show superior preparations of antigens in dimethylsulfoxide, avoiding their rapid decomposition in water (t1/2 1.5 h, 37 °C). Antigen solution structures, MAIT cell activation potencies (EC50 3–500 pM), and chemical stabilities are described. Computer analyses of antigen structures reveal stereochemical and energetic influences on MAIT cell activation, enabling design of a water stable synthetic antigen (EC50 2 nM). Like native antigens, this antigen preparation induces MR1 refolding and upregulates surface expression of human MR1, forms MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. These antigens also induce MAIT cell accumulation in mouse lungs after administration with a co-stimulant. These chemical and immunological findings provide new insights into antigen properties and MAIT cell activation.

Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth.

Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9–370 nM), with belinostat, panobinostat and vorinostat having 8–45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 μM) or S. mansoni schistosomula (IC50 > 10 μM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 μM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4–7 and 4–10 after infection (P < 0.05), respectively.

Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

Three Homology Models of PAR2 Derived from Different Templates: Application to Antagonist Discovery.

Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor (GPCR) involved in inflammation and metabolism. It is activated through cleavage of its N-terminus by proteases. The new N-terminus functions as a tethered ligand that folds back and intramolecularly activates PAR2, initiating multiple downstream signaling pathways. The only compounds reported to date to inhibit PAR2 activation are of moderate potency. Three structural models for PAR2 have been constructed based on sequence homology with known crystal structures for bovine rhodopsin, human ORL-1 (also called nociceptin/orphanin FQ receptor), and human PAR1. The three PAR2 model structures were compared and used to predict potential interactions with ligands. Virtual screening for ligands using the Chembridge database, and either ORL-1 or PAR1 derived PAR2 models led to identification of eight new small molecule PAR2 antagonists (IC50 10-100 μM). Notably, the most potent compound 1 (IC50 11 μM) was derived from the less homologous template protein, human ORL-1. The results suggest that virtual screening against multiple homology models of the same GPCR can produce structurally diverse antagonists and that this may be desirable even when some models have less sequence homology with the target protein.

Potent small agonists of protease activated receptor 2

Many proteases cut the PAR2 N-terminus resulting in conformational changes that activate cells. Synthetic peptides corresponding to newly exposed N-terminal sequences of PAR2 also activate the receptor at micromolar concentrations. PAR2-selective small molecules reported here induce PAR2-mediated intracellular calcium signaling at nanomolar concentrations (EC50 = 15-100 nM, iCa(2+), CHO-hPAR2 cells). These are the most potent and efficient small molecule ligands to activate PAR2-mediated calcium release and chemotaxis, including for human breast and prostate cancer cells.

A Potent Antagonist of Protease-Activated Receptor 2 That Inhibits Multiple Signaling Functions in Human Cancer Cells

Protease-activated receptor 2 (PAR2) is a cell surface protein linked to G-protein dependent and independent intracellular signaling pathways that produce a wide range of physiological responses, including those related to metabolism, inflammation, pain, and cancer. Certain proteases, peptides, and nonpeptides are known to potently activate PAR2. However, no effective potent PAR2 antagonists have been reported yet despite their anticipated therapeutic potential. This study investigates antagonism of key PAR2-dependent signaling properties and functions by the imidazopyridazine compound I-191 (4-(8-(tert-butyl)-6-(4-fluorophenyl)imidazo[1,2-b]pyridazine-2-carbonyl)-3,3-dimethylpiperazin-2-one) in cancer cells. At nanomolar concentrations, I-191 inhibited PAR2 binding of and activation by structurally distinct PAR2 agonists (trypsin, peptide, nonpeptide) in a concentration-dependent manner in cells of the human colon adenocarcinoma grade II cell line (HT29). I-191 potently attenuated multiple PAR2-mediated intracellular signaling pathways leading to Ca2+ release, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, Ras homologue gene family, member A (RhoA) activation, and inhibition of forskolin-induced cAMP accumulation. The mechanism of action of I-191 was investigated using binding and calcium mobilization studies in HT29 cells where I-191 was shown to be noncompetitive and a negative allosteric modulator of the agonist 2f-LIGRL-NH2. The compound alone did not activate these PAR2-mediated pathways, even at high micromolar concentrations, indicating no bias in these signaling properties. I-191 also potently inhibited PAR2-mediated downstream functional responses, including expression and secretion of inflammatory cytokines and cell apoptosis and migration, in human colon adenocarcinoma grade II cell line (HT29) and human breast adenocarcinoma cells (MDA-MB-231). These findings indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signaling pathways and functional responses. I-191 may be a valuable tool for characterizing PAR2 functions in cancer and in other cellular, physiological, and disease settings.