Warren S. Weiner

Interrogating a Hexokinase-Selected Small-Molecule Library for Inhibitors of Plasmodium falciparum Hexokinase

ABSTRACT Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (∼26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 μM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥0.6 and <6.8 μM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure–activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

A Selective ATP-Binding Cassette Subfamily G Member 2 Efflux Inhibitor Revealed via High-Throughput Flow Cytometry

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)–driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

Optimization and Evaluation of Antiparasitic Benzamidobenzoic Acids as Inhibitors of Kinetoplastid Hexokinase 1

Kinetoplastid‐based infections are neglected diseases that represent a significant human health issue. Chemotherapeutic options are limited due to toxicity, parasite susceptibility, and poor patient compliance. In response, we studied a molecular‐target‐directed approach involving intervention of hexokinase activity—a pivotal enzyme in parasite metabolism. A benzamidobenzoic acid hit with modest biochemical inhibition of Trypanosoma brucei hexokinase 1 (TbHK1, IC50=9.1 μm), low mammalian cytotoxicity (IMR90 cells, EC50>25 μm), and no appreciable activity on whole bloodstream‐form (BSF) parasites was optimized to afford a probe with improved TbHK1 potency and, significantly, efficacy against whole BSF parasites (TbHK1, IC50=0.28 μm; BSF, ED50=1.9 μm). Compounds in this series also inhibited the hexokinase enzyme from Leishmania major (LmHK1), albeit with less potency than toward TbHK1, suggesting that inhibition of the glycolytic pathway may be a promising opportunity to target multiple disease‐causing trypanosomatid protozoa.