In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth.

Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

The identification of beta-hydroxy carboxylic acids as selective MMP-12 inhibitors.

A new class of selective MMP-12 inhibitors have been identified via high throughput screening. Crystallization with MMP-12 confirmed the mode of binding and allowed initial optimization to be carried out using classical structure based design.

Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1.

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-ß-D-ribose-2'-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.

Optimization of novel acyl pyrrolidine inhibitors of hepatitis C virus RNA-dependent RNA polymerase leading to a development candidate.

Optimization of a pyrrolidine-based template using structure-based design and physicochemical considerations has provided a development candidate 20b (3082) with submicromolar potency in the HCV replicon and good pharmacokinetic properties.

Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxohexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 2. Potency and chirality.

The stereospecific synthesis of a series of alpha-methylpyrrolidine-5,5-trans-lactam inhibitors of human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series has defined the size and chirality of the alpha-substituent, optimized the acyl substituent on the lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this has led to the discovery of potent serine protease inhibitors that are highly selective for the viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase. The mechanism of action of our lead compounds has been established by mass spectrometry, and enzymatic degradation of HCMV deltaAla protease acylated with these inhibitors showed that Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained and used to model the conformationally restricted, chiral (S)-proline-alpha-methyl-5,5-trans-lactams into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this series. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the dansyl-(S)-proline alpha-methyl-5,5-trans-lactam template, which have low nanomolar activity against the viral enzyme.

Discovery of further pyrrolidine trans-lactams as inhibitors of human neutrophil elastase (HNE) with potential as development candidates and the crystal structure of HNE complexed with an inhibitor (GW475151).

Described herein is a modern approach to the rapid preparation and evaluation of compounds as potential back-up drug candidates. GW311616A, 1, a derivative of pyrrolidine trans-lactams, has previously been described as a potent, orally active inhibitor of human neutrophil elastase (HNE) for the treatment of respiratory disease. These properties made it a suitable candidate for development. Described here is the discovery of three further derivatives of pyrrolidine trans-lactams, which fulfill the criteria required for back-up candidates 28, 29, and 32. These include increased activity in inhibiting HNE in human whole blood (HWB) and comparable pharmacokinetic properties, in particular clearance, in two species. To provide a rapid assessment of clearance, cassette dosing in dog was used. Modern array techniques, including the synthesis of mixtures, were used to synthesize compounds rapidly. Having selected three potential compounds as back-up candidates, they were prepared as single enantiomers and profiled in in vitro and in vivo assays and evaluated pharmacokinetically in rat and dog. These compounds are highly potent and selective HNE inhibitors, with a prolonged pharmacodynamic action. Pharmacokinetically, these compounds are comparable with 1 while they are more potent in HWB. Compound 28, however, has a higher clearance. One of these compounds, 32, was cocrystallized with HNE, and features of this structure are described and compared with the cocrystal structure of 1 in porcine pancreatic elastase.

Crystallizing Membrane Proteins in the Lipidic Mesophase. Experience with Human Prostaglandin E2 Synthase 1 and an Evolving Strategy.

The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic α-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des.2013, 14, 2846-2857). Here, we describe its application to the human α-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 °C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method.

Identification of a Novel and Selective Series of Itk Inhibitors via a Template-Hopping Strategy.

Inhibition of Itk potentially constitutes a novel, nonsteroidal treatment for asthma and other T-cell mediated diseases. In-house kinase cross-screening resulted in the identification of an aminopyrazole-based series of Itk inhibitors. Initial work on this series highlighted selectivity issues with several other kinases, particularly AurA and AurB. A template-hopping strategy was used to identify a series of aminobenzothiazole Itk inhibitors, which utilized an inherently more selective hinge binding motif. Crystallography and modeling were used to rationalize the observed selectivity. Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays.