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Galectin proteins have been targets of interest in numerous therapeutic areas for some time. Galectin-3 has been identified as a target of particular interest because of its unique structural characteristics and physiological profile. Recent literature indicates Gal-3 inhibition can decrease myofibroblast activation and procollagen expression with the potential to affect the progression of fibrosis in the lung, liver and kidney. Potential π-stacking interactions between one monosaccharide ligand bound to the carbohydrate recognition domain of a galectin-3 protein and a second ligand bound to a different galectin-3 protein molecule were observed in the extended crystalline lattice in an X-ray crystal structure obtained while studying the monosaccharide structure-activity relationship. The direct interaction between the ligands suggests a potential for dimeric galectin-3 inhibitors which bind to two galectin-3 molecules simultaneously. This work describes the exploration of dimers designed to further probe these observations and explore the potential of binding to dimeric or higher order multimeric galectin-3 assemblies.

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

We describe an integrated approach of using hydrogen–deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the bindi...

Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen-deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking. HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the C'D-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.

GSK3532795 (formerly BMS955176) is a second-generation maturation inhibitor (MI) that progressed through a Phase 2b study for treatment of HIV-1 infection. Resistance development to GSK3532795 was evaluated through in vitro methods and was correlated with information obtained in a Phase 2a proof-of-concept study in HIV-1 infected participants. Both low and high concentrations of GSK3532795 were used for selections in vitro, and reduced susceptibility to GSK3532795 mapped specifically to amino acids near the capsid/ spacer peptide 1 (SP1) junction, the cleavage of which is blocked by MIs. Two key substitutions, A364V or V362I, were selected, the latter requiring secondary substitutions to reduce susceptibility to GSK3532795. Three main types of secondary substitutions were observed, none of which reduced GSK3532795 susceptibility in isolation. The first type was in the capsid C-terminal domain and downstream SP1 region (including (Gag numbering) R286K, A326T, T332S/N, I333V and V370A/M). The second, was an R41G substitution in viral protease that occurred with V362I. The third was seen in the capsid N-terminal domain, within the cyclophilin A binding domain (V218A/M, H219Q and G221E). H219Q increased viral replication capacity and reduced susceptibility of poorly growing viruses. In the Phase 2a study, a subset of these substitutions was also observed at baseline and some were selected following GSK35323795 treatment in HIV-1-infected participants.

HIV-1 maturation inhibition (MI) has been clinically validated as an approach to the control of HIV-1 infection. However, identifying an MI with both broad polymorphic spectrum coverage and good oral exposure has been challenging. Herein, we describe the design, synthesis, and preclinical characterization of a potent, orally active, second generation HIV-1 MI, BMS-955176 (2), which is currently in Phase IIb clinical trials as part of a combination antiretroviral regimen.

[Image: see text] In solving the P-gp and BCRP transporter-mediated efflux issue in a series of benzofuran-derived pan-genotypic palm site inhibitors of the hepatitis C virus NS5B replicase, it was found that close attention to physicochemical properties was essential. In these compounds, where both molecular weight (MW >579) and TPSA (>110 Å(2)) were high, attenuation of polar surface area together with weakening of hydrogen bond acceptor strength of the molecule provided a higher intrinsic membrane permeability and more desirable Caco-2 parameters, as demonstrated by trifluoroacetamide 11 and the benchmark N-ethylamino analog 12. In addition, the tendency of these inhibitors to form intramolecular hydrogen bonds potentially contributes favorably to the improved membrane permeability and absorption. The functional group minimization that resolved the efflux problem simultaneously maintained potent inhibitory activity toward a gt-2 HCV replicon due to a switching of the role of substituents in interacting with the Gln414 binding pocket, as observed in gt-2a NS5B/inhibitor complex cocrystal structures, thus increasing the efficiency of the optimization. Noteworthy, a novel intermolecular S=O···C=O n → π* type interaction between the ligand sulfonamide oxygen atom and the carbonyl moiety of the side chain of Gln414 was observed. The insights from these structure–property studies and crystallography information provided a direction for optimization in a campaign to identify second generation pan-genotypic NS5B inhibitors.

Higher order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP) and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs. backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 ∼ 6.2 pM vs. Kd, Adenctin2 ∼ 46 nM), and evaluate the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adenctin1 and adenctin2 binding, is a flexible loop that connects two β-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with similar apparent biophysical properties.

The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B polymerase via binding to the primer grip site is presented. Many challenges, including poor oral bioavailability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered and overcome through SAR studies. This work culminated in the selection of BMS-986139 (43) as a preclinical candidate.

Alkoxyanthranilic acid derivatives have been identified to inhibit HCV NS5B polymerase, binding in an allosteric site located at the convergence of the palm and thumb regions. Information from co-crystal structures guided the structural design strategy. Ultimately, two independent structural modifications led to a similar shift in binding mode that when combined led to a synergistic improvement in potency and the identification of inhibitors with sub-micromolar HCV NS5B binding potency.

A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

New applications of fingerprints of multiple potential 4-point three-dimensional (3D) pharmacophores in combinatorial library design and virtual screening are presented. Preliminary results demonstrating the feasibility of a simulated annealing process for combinatorial reagent selection that concurrently optimizes product diversity in BCUT chemistry space and in terms of unique 4-point pharmacophores are discussed, and the advantage of using a customized chemistry-space derived for the library design is demonstrated. In addition, an extension to the multiple pharmacophore method for structure-based design that uses the shape of the target site as an additional constraint is presented. This development enables the docking process to be quantified in terms of the number and identities of the pharmacophoric hypotheses that can be matched by a compound or a library of compounds. The design of an example combinatorial library based on the Ugi condensation reaction and a serine protease active site is described.

Retro-cycloadditions and thermal rearrangements of hydrocarbons have been investigated with density functional theory (B3LYP) and complete active space multiconfiguration self consistent field theory (CASSCF and CASPT2). We review recent results from our laboratories. Subjects are the laser-induced retro-Diels- Alder reactions of norbornene, thermal retro-Diels-Alder reactions of norbornenes and isopropylidenorbornene, 1,3-sigmatropic shifts of vinylcyclopropane and bicyclo(3.2.0)heptenes, and the 1,5-sigmatropic shifts of norcaradienes. The competition between concerted and stepwise processes and the nature of diradicals formed in these processes are described.

A highly specific Diels-Alder protein catalyst was made by manipulating the antibody repertoire of the immune system. The catalytic antibody 13G5 catalyzes a disfavored exo Diels-Alder transformation in a reaction for which there is no natural enzyme counterpart and that yields a single regioisomer in high enantiomeric excess. The crystal structure of the antibody Fab in complex with a ferrocenyl inhibitor containing the essential haptenic core that elicited 13G5 was determined at 1.95 angstrom resolution. Three key antibody residues appear to be responsible for the observed catalysis and product control. Tyrosine-L36 acts as a Lewis acid activating the dienophile for nucleophilic attack, and asparagine-L91 and aspartic acid–H50 form hydrogen bonds to the carboxylate side chain that substitutes for the carbamate diene substrate. This hydrogen-bonding scheme leads to rate acceleration and also pronounced stereoselectivity. Docking experiments with the four possible ortho transition states of the reaction explain the specific exo effect and suggest that the (3 R ,4 R )- exo stereoisomer is the preferred product.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

The reaction of dicyanoacetylene (DCA) with the partially hydrogenated dimethano-bridged anthracene and pentacene derivs. I and II lead to the (1:1) Diels - Alder adducts III and IV, resp., and to the 9,10-dihydronaphthalene derivs. V and VI, resp. These compds. are intensely colored, most likely due to a charge-transfer absorption. Photolysis of the (1:1) adducts III and IV produces the corresponding cyclooctatetraene derivs. , which are not planar despite the torsional constraints caused by the fusion of the eight-membered ring to norbornane and norbornene units. The mechanisms of formation of the dihydronaphthalene derivs. V and VI were elucidated by the use of high pressure; this allowed the (2:1) adducts VII and VIII to be detected as intermediates in the reaction of DCA with III or IV to give V and VI, resp. A degenerate rearrangement consisting formally of a [1,5] vinyl shift in V or VI could be detected by their temp.-dependent 1H NMR spectra. The mechanisms of the [1,5]-sigmatropic shift reactions in V and IX have been examd. at the Becke3LYP/6-31G* level of theory. Stepwise mechanisms are predicted, and the calcd. activation barrier is in good agreement with the measured DH.thermod.. The finding that the irreversible rearrangement of V accompanied by the elimination of HCN to give the azulene deriv. proceeds only on heating of V to 80 Deg in a polar solvent is good evidence for a polar mechanism in this case.

Catalytic antibodies that control the reaction pathways of the Diels-Alder cycloaddition have been generated. One antibody catalyzes the favored endo and the other the disfavored exo pathway to yield the respective cis and trans adducts in enantiomerically pure form. A comparison of the x-ray structure of the hapten with the calculated geometry of the transition structure showed that [2.2.2] bicyclic compounds are excellent mimics of the transition state of the Diels-Alder reaction. To achieve catalysis and the high degree of stereoselectivity shown here, the antibody must simultaneously control the conformation of the individual reactants and their relation to each other. In the case of the disfavored process, binding energy must be used to reroute the reaction along a higher energy pathway. The rerouting of reaction pathways has become a major focus of antibody catalysis and other disfavored reactions can be expected to be catalyzed so long as the energy barrier is not extreme. The energy requirements needed for absolute control of all of the stereoisomers of many Diels-Alder reactions fall in the energy range (approximately 20 kilocalories per mole) deliverable by antibody binding.

2 H NMR, EPR, computational and product analyses of the photolysis of 2,4-diphenylpentan-3-one (DPP) adsorbed on MFI size/shape selective zeolites are consistent with supra- molecular structural changes as a function of surface coverage that provide a novel method for the generation of persistent diffusing organic free radicals. The MFI topology zeolites (e.g. silicalite and ZSM-5) are of technical significance in both size/shape selective catalysis and in molecular sieve separations. 1 The technically attractive properties of MFI zeolites originate from supramolecular, structural (substrates as guests, zeolite crystal as host) and dynamic (shape/size dependent supramolecular molecular dif- fusion) characteristics, i.e. substrates possess 'effective' molec- ular diameters comparable to the size/shape of the holes on the external surface of the zeolite crystal and/or the channels and intersections on the internal surface. 2 The siting and diffusion of a substrate are vital in determining the product selectivity in catalysis and the resolution efficiency in separations. 3 Understanding the role of diffusion and siting requires the uncoupling of both supramolecular, structural and dynamic features from the effects of activation at active sites and rearrangements of reactive intermediates. The effects of activation are pronounced at high temperatures with zeolites possessing strongly acidic sites ( e.g. HZSM-5). We report a supramolecular photochemical investigation 4 at room temperature in which the activation and diffusional/siting effects of zeolite catalysis are uncoupled. Activation of the substrate towards reaction is provided by photochemical methods which (i) excite adsorbed substrates whose sitings and reaction products are directly determined by 2H NMR analysis and (ii) produce persistent 5,6 adsorbed carbon-centred radical intermediates whose structure and mobility are directly deter- mined by EPR spectroscopy. Computational methods are employed for (i) analysis of the plausibility of the siting and diffusional processes on the external and internal surfaces, (ii) rationalization of the variation of product distribution as a function of supramolecular structure, and (iii) simulation of the EPR spectra of the persistent supramolecular radical species formed upon photoexcitation of the adsorbed substrates. In the molecular (solution) photochemistry (eqns. (1)-(3)) of 2,4-diphenylpentan-3-one (DPP) a-cleavage from T1 produces, after rapid loss of CO, a-methylbenzyl radicals (MB·) (eqn. (1)), which undergo random radical combination to produce 2,3-diphenylbutane (DPB) (ca. 95%) and disproportionates to styrene (S) and ethylbenzene (EB) (ca. 5%). The rates of the radical-radical reactions in eqns. (2) and (3) are close to diffusion controlled 5,8 and, in the absence of radical scavengers, determine the lifetime of the 'molecular' radicals MB ·. Results of product analysis and EPR measurements as a function of coverage (DPP/(Na)ZSM-5 (w/w), Si/Al = 80, average particle size ca. 0.1 m) showed that (i) the dominant products depend on the coverage, with disproportionation to form S and EB being favored by low coverage ( < 0.3%) and combination to form DPB being increasingly favored as the coverage increases,9 and (ii) the maximum EPR intensity of the signal of the persistent MB·radicals is insensitive to loading. For example, the ratio of disproportionation to combination of MB·radicals in solution is typically of the order of 0.1; at 0.3% loading of DPP the ratio is 3.1.