Pyrone-based inhibitors of metalloproteinase types 2 and 3 may work as conformation-selective inhibitors.

Matrix metalloproteinases are zinc-containing enzymes capable of degrading all components of the extracellular matrix. Owing to their role in human disease, matrix metalloproteinase have been the subject of extensive study. A bioinorganic approach was recently used to identify novel inhibitors based on a maltol zinc-binding group, but accompanying molecular-docking studies failed to explain why one of these inhibitors, AM-6, had approximately 2500-fold selectivity for MMP-3 over MMP-2. A number of studies have suggested that the matrix-metalloproteinase active site is highly flexible, leading some to speculate that differences in active-site flexibility may explain inhibitor selectivity. To extend the bioinorganic approach in a way that accounts for MMP-2 and MMP-3 dynamics, we here investigate the predicted binding modes and energies of AM-6 docked into multiple structures extracted from matrix-metalloproteinase molecular dynamics simulations. Our findings suggest that accounting for protein dynamics is essential for the accurate prediction of binding affinity and selectivity. Additionally, AM-6 and other similar inhibitors likely select for and stabilize only a subpopulation of all matrix-metalloproteinase conformations sampled by the apo protein. Consequently, when attempting to predict ligand affinity and selectivity using an ensemble of protein structures, it may be wise to disregard protein conformations that cannot accommodate the ligand

Mapping the druggable allosteric space of G-protein coupled receptors: a fragment-based molecular dynamics approach.

To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the beta1 (beta1AR) and beta2 (beta2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify 'hot spots' with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 micros. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies

Lapatinib-binding protein kinases in the African trypanosome: identification of cellular targets for kinase-directed chemical scaffolds.

Human African trypanosomiasis is caused by the eukaryotic microbe Trypanosoma brucei. To discover new drugs against the disease, one may use drugs in the clinic for other indications whose chemical scaffolds can be optimized via a medicinal chemistry campaign to achieve greater potency against the trypanosome. Towards this goal, we tested inhibitors of human EGFR and/or VEGFR as possible anti-trypanosome compounds. The 4-anilinoquinazolines canertinib and lapatinib, and the pyrrolopyrimidine AEE788 killed bloodstream T. brucei in vitro with GI(50) in the low micromolar range. Curiously, the genome of T. brucei does not encode EGFR or VEGFR, indicating that the drugs recognize alternate proteins. To discover these novel targets, a trypanosome lysate was adsorbed to an ATP-sepharose matrix and washed with a high salt solution followed by nicotinamide adenine dinucleotide (NAD(+)). Proteins that remained bound to the column were eluted with drugs, and identified by mass spectrometry/bioinformatics. Lapatinib bound to Tb927.4.5180 (termed T. brucei lapatinib-binding protein kinase-1 (TbLBPK1)) while AEE788 bound Tb927.5.800 (TbLBPK2). When the NAD(+) wash was omitted from the protocol, AEE788, canertinib and lapatinib eluted TbLBPK1, TbLBPK2, and Tb927.3.1570 (TbLBPK3). In addition, both canertinib and lapatinib eluted Tb10.60.3140 (TbLBPK4), whereas only canertinib desorbed Tb10.61.1880 (TbCBPK1). Lapatinib binds to a unique conformation of protein kinases. To gain insight into the structural basis for lapatinib interaction with TbLBPKs, we constructed three-dimensional models of lapatinib•TbLBPK complexes, which confirmed that TbLBPKs can adopt lapatinib-compatible conformations. Further, lapatinib, AEE788, and canertinib were docked to TbLBPKs with favorable scores. Our studies (a) present novel targets of kinase-directed drugs in the trypanosome, and (b) offer the 4-anilinoquinazoline and pyrrolopyrimidines as scaffolds worthy of medicinal chemistry and structural biology campaigns to develop them into anti-trypanosome drugs

Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes

Centrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics

A single amino acid determines preference between phospholipids and reveals length restriction for activation ofthe S1P₄ receptor

Background<br/><br/> Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P<sub>1</sub> from S1P to 18:1 LPA.<br/><br/> Results<br/><br/> We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P<sub>4</sub>. This mutation severely affected the response of S1P<sub>4</sub> to S1P in a [<sup>35</sup>S]GTPγS binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P<sub>4</sub> receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors.<br/><br/> Conclusions<br/><br/> Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P<sub>1</sub> receptor

New insights on repellent recognition by <i>Anopheles gambiae</i> odorant-binding protein 1

It is generally recognized that insect odorant binding proteins (OBPs) mediate the solubilisation and transport of hydrophobic odorant molecules and contribute to the sensitivity of the insect olfactory system. However, the exact mechanism by which OBPs deliver odorants to olfactory receptors and their role, if any, as selectivity filters for specific odorants, are still a matter of debate. In the case of Anopheles gambiae recent studies indicate that ligand discrimination is effected through the formation of heterodimers such as AgamOBP1 and AgamOBP4 (odorant binding proteins 1 and 4 from Anopheles gambiae). Furthermore, AgamOBPs have been reported to be promiscuous in binding more than one ligand simultaneously and repellents such as DEET (N,N-diethyl-3-toluamide) and 6-MH (6-methyl-5-hepten-2-one) interact directly with mosquito OBPs and/or compete for the binding of attractive odorants thus disrupting OBP heterodimerisation. In this paper, we propose mechanisms of action of DEET and 6-MH. We also predict that ligand binding can occur in several locations of AgamOBP1 with partial occupancies and propose structural features appropriate for repellent pharmacophores

Binding mode analyses of NAP derivatives as mu opioid receptor selective ligands through docking studies and molecular dynamics simulation

Mu opioid receptor selective antagonists are highly desirable because of their utility as pharmacological probes for receptor characterization and functional studies. Furthermore, the mu opioid receptors act as an important target in drug abuse and addiction treatment. Previously, we reported NAP as a novel lead compound with high selectivity and affinity towards the mu opioid receptor. Based on NAP, we have synthesized its derivatives and further characterized their binding affinities and selectivity towards the receptor. NMP and NGP were identified as the two most selective MOR ligands among NAP derivatives. In the present study, molecular modeling methods were applied to assess the dual binding modes of NAP derivatives, particularly on NMP and NGP, in three opioid receptors, in order to analyze the effects of structural modifications on the pyridyl ring of NAP on the binding affinity and selectivity. The results indicated that the steric hindrance, electrostatic, and hydrophobic effects caused by the substituents on the pyridyl ring of NAP contributed complimentarily on the binding affinity and selectivity of NAP derivatives to three opioid receptors. Analyses of these contributions provided insights on future design of more potent and selective mu opioid receptor ligands

Mining electron density for functionally relevant protein polysterism in crystal structures.

This review focuses on conceptual and methodological advances in our understanding and characterization of the conformational heterogeneity of proteins. Focusing on X-ray crystallography, we describe how polysterism, the interconversion of pre-existing conformational substates, has traditionally been analyzed by comparing independent crystal structures or multiple chains within a single crystal asymmetric unit. In contrast, recent studies have focused on mining electron density maps to reveal previously 'hidden' minor conformational substates. Functional tests of the importance of minor states suggest that evolutionary selection shapes the entire conformational landscape, including uniquely configured conformational substates, the relative distribution of these substates, and the speed at which the protein can interconvert between them. An increased focus on polysterism may shape the way protein structure and function is studied in the coming years