Justin R. Harrison

Discovery of a novel class of orally active trypanocidal N-myristoyltransferase inhibitors.

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.

Chemical Validation of Trypanothione Synthetase A POTENTIAL DRUG TARGET FOR HUMAN TRYPANOSOMIASIS

In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 × EC50 for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.

Design, Synthesis and Biological Evaluation of Trypanosoma brucei Trypanothione Synthetase Inhibitors

Trypanothione synthetase (TryS) is essential for the survival of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis. It is one of only a handful of chemically validated targets for T. brucei in vivo. To identify novel inhibitors of TbTryS we screened our in‐house diverse compound library that contains 62 000 compounds. This resulted in the identification of six novel hit series of TbTryS inhibitors. Herein we describe the SAR exploration of these hit series, which gave rise to one common series with potency against the enzyme target. Cellular studies on these inhibitors confirmed on‐target activity, and the compounds have proven to be very useful tools for further study of the trypanothione pathway in kinetoplastids.

Lead optimization of a pyrazole sulfonamide series of Trypanosoma brucei N-myristoyltransferase inhibitors: identification and evaluation of CNS penetrant compounds as potential treatments for stage 2 human African trypanosomiasis.

Trypanosoma bruceiN-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood–brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT.

N-Myristoyltransferase Is a Cell Wall Target in Aspergillus fumigatus

Treatment of filamentous fungal infections relies on a limited repertoire of antifungal agents. Compounds possessing novel modes of action are urgently required. N-myristoylation is a ubiquitous modification of eukaryotic proteins. The enzyme N-myristoyltransferase (NMT) has been considered a potential therapeutic target in protozoa and yeasts. Here, we show that the filamentous fungal pathogen Aspergillus fumigatus possesses an active NMT enzyme that is essential for survival. Surprisingly, partial repression of the gene revealed downstream effects of N-myristoylation on cell wall morphology. Screening a library of inhibitors led to the discovery of a pyrazole sulphonamide compound that inhibits the enzyme and is fungicidal under partially repressive nmt conditions. Together with a crystallographic complex showing the inhibitor binding in the peptide substrate pocket, we provide evidence of NMT being a potential drug target in A. fumigatus.

A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi–bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP–inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.

Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of Trypanosoma cruzi

Background Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT) has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids. Methodology/Principal Findings Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8) have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors. Conclusions/Significance Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy.

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.

A Molecular Hybridization Approach for the Design of Potent, Highly Selective, and Brain-Penetrant N-Myristoyltransferase Inhibitors.

Crystallography has guided the hybridization of two series of Trypanosoma bruceiN-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood–brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease.