Peter Ray

Respiratory Flexibility in Response to Inhibition of Cytochrome c Oxidase in Mycobacterium tuberculosis

ABSTRACT We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs.

Prediction of Drug Penetration in Tuberculosis Lesions.

The penetration of antibiotics in necrotic tuberculosis lesions is heterogeneous and drug-specific, but the factors underlying such differential partitioning are unknown. We hypothesized that drug binding to macromolecules in necrotic foci (or caseum) prevents passive drug diffusion through avascular caseum, a critical site of infection. Using a caseum binding assay and MALDI mass spectrometry imaging of tuberculosis drugs, we showed that binding to caseum inversely correlates with passive diffusion into the necrotic core. We developed a high-throughput assay relying on rapid equilibrium dialysis and a caseum surrogate designed to mimic the composition of native caseum. A set of 279 compounds was profiled in this assay to generate a large data set and explore the physicochemical drivers of free diffusion into caseum. Principle component analysis and modeling of the data set delivered an in silico signature predictive of caseum binding, combining 69 molecular descriptors. Among the major positive drivers of binding were high lipophilicity and poor solubility. Determinants of molecular shape such as the number of rings, particularly aromatic rings, number of sp(2) carbon counts, and volume-to-surface ratio negatively correlated with the free fraction, indicating that low-molecular-weight nonflat compounds are more likely to exhibit low caseum binding properties and diffuse effectively through caseum. To provide simple guidance in the property-based design of new compounds, a rule of thumb was derived whereby the sum of the hydrophobicity (clogP) and aromatic ring count is proportional to caseum binding. These tools can be used to ensure desirable lesion partitioning and guide the selection of optimal regimens against tuberculosis.

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.

Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors

Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.

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.

2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure–Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

Mycobacterium tuberculosis (MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.

Identification of Morpholino Thiophenes as Novel Mycobacterium tuberculosis Inhibitors, Targeting QcrB

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino–thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure–activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.

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.

Correction for Arora et al., “Respiratory Flexibility in Response to Inhibition of Cytochrome c Oxidase in Mycobacterium tuberculosis”

Volume 58, no. 11, p. 6962–6965, 2014, [https://doi.org/10.1128/AAC.03486-14][1]. Page 6963, left column, second paragraph: the following text in lines 5 to 10 should be deleted. “We deleted this oxidase in H37Rv by replacing a 221-bp MluI fragment in the cydABDC operon with the aph gene