Lead Optimization of 3,5-Disubstituted-7-Azaindoles for the Treatment of Human African Trypanosomiasis

Neglected tropical diseases such as human African trypanosomiasis (HAT) are prevalent primarily in tropical climates and among populations living in poverty. Historically, the lack of economic incentive to develop new treatments for these diseases has meant that existing therapeutics have serious shortcomings in terms of safety, efficacy, and administration, and better therapeutics are needed. We now report a series of 3,5-disubstituted-7-azaindoles identified as growth inhibitors of Trypanosoma brucei, the parasite that causes HAT, through a high-throughput screen. We describe the hit-to-lead optimization of this series and the development and preclinical investigation of 29d, a potent antitrypanosomal compound with promising pharmacokinetic (PK) parameters. This compound was ultimately not progressed beyond in vivo PK studies due to its inability to penetrate the blood-brain barrier (BBB), critical for stage 2 HAT treatments

Benznidazole biotransformation and multiple targets in <i>Trypanosoma</i> cruzi revealed by metabolomics

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methodology/Principal findings&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions/significance&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi

Analysis and Prediction of Translation Rate Based on Sequence and Functional Features of the mRNA

Protein concentrations depend not only on the mRNA level, but also on the translation rate and the degradation rate. Prediction of mRNA's translation rate would provide valuable information for in-depth understanding of the translation mechanism and dynamic proteome. In this study, we developed a new computational model to predict the translation rate, featured by (1) integrating various sequence-derived and functional features, (2) applying the maximum relevance & minimum redundancy method and incremental feature selection to select features to optimize the prediction model, and (3) being able to predict the translation rate of RNA into high or low translation rate category. The prediction accuracies under rich and starvation condition were 68.8% and 70.0%, respectively, evaluated by jackknife cross-validation. It was found that the following features were correlated with translation rate: codon usage frequency, some gene ontology enrichment scores, number of RNA binding proteins known to bind its mRNA product, coding sequence length, protein abundance and 5′UTR free energy. These findings might provide useful information for understanding the mechanisms of translation and dynamic proteome. Our translation rate prediction model might become a high throughput tool for annotating the translation rate of mRNAs in large-scale

Evaluation of a class of isatinoids identified from a high-throughput screen of human kinase inhibitors as anti-Sleeping Sickness agents

New treatments are needed for neglected tropical diseases (NTDs) such as Human African trypanosomiasis (HAT), Chagas disease, and schistosomiasis. Through a whole organism high-throughput screening campaign, we previously identified 797 human kinase inhibitors that grouped into 59 structural clusters and showed activity against T. brucei, the causative agent of HAT. We herein report the results of further investigation of one of these clusters consisting of substituted isatin derivatives, focusing on establishing structure-activity and -property relationship scope. We also describe their in vitro absorption, distribution, metabolism, and excretion (ADME) properties. For one isatin, NEU-4391, which offered the best activity-property profile, pharmacokinetic parameters were measured in mice

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug clas

Mutagenesis and Functional Studies with Succinate Dehydrogenase Inhibitors in the Wheat Pathogen Mycosphaerella graminicola

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness

Exploring the Trypanosoma brucei Hsp83 Potential as a Target for Structure Guided Drug Design

Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs--whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases. In this paper, we report the exploration of T. brucei Hsp83--a homolog of human Hsp90--as a drug target using multiple biophysical and biochemical techniques. Our approach included the characterization of the chemical sensitivity of the parasitic chaperone against a library of known Hsp90 inhibitors by means of differential scanning fluorimetry (DSF). Several compounds identified by this screening procedure were further studied using isothermal titration calorimetry (ITC) and X-ray crystallography, as well as tested in parasite growth inhibitions assays. These experiments led us to the identification of a benzamide derivative compound capable of interacting with TbHsp83 more strongly than with its human homologs and structural rationalization of this selectivity. The results highlight the opportunities created by subtle structural differences to develop new series of compounds to selectively target the Trypanosoma brucei chaperone and effectively kill the sleeping sickness parasite

Identification of cinnabarinic acid as a novel endogenous aryl hydrocarbon receptor ligand that drives IL-22 production

The aryl hydrocarbon receptor (AHR) binds to environmental toxicants including synthetic halogenated aromatic hydrocarbons and is involved in a diverse array of biological processes. Recently, the AHR was shown to control host immunity by affecting the ba

Selectivity and Physicochemical Optimization of Repurposed Pyrazolo[1,5-b]pyridazines for the Treatment of Human African Trypanosomiasis

From a high-throughput screen of 42 444 knownhuman kinases inhibitors, a pyrazolo[1,5-b]pyridazine scaffold wasidentified to begin optimization for the treatment of human Africantrypanosomiasis. Previously reported data for analogous com-pounds against human kinases GSK-3β, CDK-2, and CDK-4 wereleveraged to try to improve the selectivity of the series, resulting in23awhich showed selectivity forT. b. bruceiover these threehuman enzymes. In parallel, properties known to influence theabsorption, distribution, metabolism, and excretion (ADME)profile of the series were optimized resulting in20gbeingprogressed into an efficacy study in mice. Though20gshowedtoxicity in mice, it also demonstrated CNS penetration in a PKstudy and significant reduction of parasitemia in four out of the sixmice.This work was supported by National Institutes of HealthGrants (R01AI114685 (M.P.P. and M.N.), R01AI082577(M.P.P.), R56AI099476 (M.P.P.), R01AI124046 (M.P.P.),R21AI127594 (M.P.P.), the Spanish Ministerio de Economía,Industria y Competitividad (M.N., Grant SAF2015-71444-P;D.G.P., Grant SAF2016-79957-R., and Subdirección Generalde Redes y Centros de Investigación Cooperativa (RICET)(M.N., Grant RD16/0027/0019; D.G.P., Grant RD16/0027/0014), Grant RTI2018-097210-B-100 (MINCIU-FEDER) toF.G. C.R.C. acknowledges grant support from the NIH-NIAID(Grant R21AI126296) and the Bill and Melinda GatesFoundation (Grant OPP1171488), as well as the technicalassistance of Brian M. Suzuki for screening adultS. mansoni.We are grateful to AstraZeneca for performing thein vitroADME experiments presented throughout and to CharlesRiver Labs for thein vitroADME data presented inTables S3−S5 in the Supporting Information. We thank GSK Tres Cantosopen lab foundation for running the PK studies discussed inthis publication. An academic license for ChemAxon (https://www.chemaxon.com) is gratefully acknowledged. We thankDr. Melissa Buskes for help in the preparation of thismanuscri