Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

Assessment of the drugability of initial malaria infection through miniaturized sporozoite assays and high-throughput screening.

The sporozoite stages of malaria parasites are the primary cause of infection of the vertebrate host and are targeted by (experimental) vaccines. Yet, little is known about their susceptibility to chemical intervention. Phenotypic high-throughput screens have not been feasible due to a lack of in vitro systems. Here we tested 78 marketed and experimental antimalarial compounds in miniaturized assays addressing sporozoite viability, gliding motility, hepatocyte traversal, and intrahepatocytic schizogony. None potently interfered with sporozoite viability or motility but ten compounds acted at the level of schizogony with IC50s < 100 nM. To identify compounds directly targeting sporozoites, we screened 81,000 compounds from the Global Health Diversity and reFRAME libraries in a sporozoite viability assay using a parasite expressing a luciferase reporter driven by the circumsporozoite promoter. The ionophore gramicidin emerged as the single hit from this screening campaign. Its effect on sporozoite viability translated into reduced gliding motility and an inability of sporozoites to invade human primary hepatocytes and develop into hepatic schizonts. While providing proof of concept for a small molecule sporontocidal mode of action, our combined data indicate that liver schizogony is more accessible to chemical intervention by (candidate) antimalarials.

Exhaustive sampling of the fragment space associated to a molecule leading to the generation of conserved fragments.

The first step in hit optimization is the identification of the pharmacophore, which is normally achieved by deconstruction of the hit molecule to generate "deletion analogues." In silico fragmentation approaches often focus on the generation of small fragments that do not describe properly the fragment space associated to the deletion analogues. We present significant modifications to the molecular fragmentation programme molBLOCKS, which allows the exhaustive sampling of the fragment space associated with a molecule to generate all possible molecular fragments. This generates larger fragments, by combining the smallest fragments. Additionally, it has been modified to deal with the problem of changing pharmacophoric properties through fragmentation, by highlighting bond cuts. The modified molBLOCKS programme was used on a set of drug compounds, where it generated more unique fragments than standard fragmentation approaches by increasing the number of fragments derived per compound. This fragment set was found to be more diverse than those generated by standard fragmentation programmes and was relevant to drug discovery as it contains the key fragments representing the pharmacophoric elements associated with ligand recognition. The use of dummy atoms to highlight bond cuts further increases the information content of fragments by visualizing their previous bonding pattern.

Fragment library design, synthesis and expansion: nurturing a synthesis and training platform.

The availability of suitable diverse fragment- and lead-oriented screening compounds is key for the identification of suitable chemical starting points for drug discovery programs. The physicochemical properties of molecules are crucial in determining the success of small molecules in clinical development, yet reports suggest that pharmaceutical and academic sectors often produce molecules with poor drug-like properties. We present a platform to design novel, high quality and diverse fragment- and lead-oriented libraries with appropriate physicochemical properties in a cost-efficient manner. This approach has the potential to assist the way libraries are constructed by significantly addressing the historical uneven exploration of chemical space for drug discovery. Additionally, this platform can teach undergraduates and graduates about compound library design.

De novo design of protein kinase inhibitors by in silico identification of hinge region-binding fragments.

Protein kinases constitute an attractive family of enzyme targets with high relevance to cell and disease biology. Small molecule inhibitors are powerful tools to dissect and elucidate the function of kinases in chemical biology research and to serve as potential starting points for drug discovery. However, the discovery and development of novel inhibitors remains challenging. Here, we describe a structure-based de novo design approach that generates novel, hinge-binding fragments that are synthetically feasible and can be elaborated to small molecule libraries. Starting from commercially available compounds, core fragments were extracted, filtered for pharmacophoric properties compatible with hinge-region binding, and docked into a panel of protein kinases. Fragments with a high consensus score were subsequently short-listed for synthesis. Application of this strategy led to a number of core fragments with no previously reported activity against kinases. Small libraries around the core fragments were synthesized, and representative compounds were tested against a large panel of protein kinases and subjected to co-crystallization experiments. Each of the tested compounds was active against at least one kinase, but not all kinases in the panel were inhibited. A number of compounds showed high ligand efficiencies for therapeutically relevant kinases; among them were MAPKAP-K3, SRPK1, SGK1, TAK1, and GCK for which only few inhibitors are reported in the literature.

Optimisation of pharmacokinetic properties to afford an orally bioavailable and selective V1A receptor antagonist.

The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.

Structure based evolution of a novel series of positive modulators of the AMPA receptor.

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19.

Discovery and optimisation of a selective non-steroidal glucocorticoid receptor antagonist.

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

A novel series of positive modulators of the AMPA receptor: discovery and structure based hit-to-lead studies.

Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor.

A novel series of positive modulators of the AMPA receptor: structure-based lead optimization.

Starting from lead compound 1, we demonstrate how X-ray structural data can be used to understand SAR and expediently optimize bioavailability in a novel series of AMPA receptor modulators, furnishing 5 with improved bioavailability and robust in vivo activity.