ABSTRACT
While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.
Subject(s)
Cytostatic Agents , Trypanocidal Agents , Trypanosoma brucei brucei , Trypanosomiasis, African , Animals , Humans , Trypanosomiasis, African/drug therapy , Trypanocidal Agents/therapeutic use , Trypanocidal Agents/pharmacokinetics , Cytostatic Agents/therapeutic use , Blood-Brain BarrierABSTRACT
Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25â µm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for inâ vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.
Subject(s)
Acetamides/pharmacology , Antimalarials/pharmacology , Acetamides/chemical synthesis , Acetamides/pharmacokinetics , Animals , Antimalarials/chemical synthesis , Antimalarials/pharmacokinetics , Humans , Mice , Microsomes, Liver/metabolism , Molecular Structure , Parasitic Sensitivity Tests , Plasmodium berghei/drug effects , Plasmodium cynomolgi/drug effects , Plasmodium falciparum/drug effects , Structure-Activity RelationshipABSTRACT
Crystallography has guided the hybridization of two series of Trypanosoma brucei N-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.
Subject(s)
Acyltransferases/antagonists & inhibitors , Blood-Brain Barrier/drug effects , Drug Design , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosomiasis, African/drug therapy , Animals , Cell Survival , Female , Humans , Mice , Mice, Inbred Strains , Models, Molecular , Molecular Structure , Protein Conformation , Structure-Activity Relationship , Trypanosomiasis, African/microbiologyABSTRACT
N-Myristoyltransferase (NMT) represents a promising drug target within the parasitic protozoa Trypanosoma brucei (T. brucei), the causative agent for human African trypanosomiasis (HAT) or sleeping sickness. We have previously validated T. brucei NMT as a promising druggable target for the treatment of HAT in both stages 1 and 2 of the disease. We report on the use of the previously reported DDD85646 (1) as a starting point for the design of a class of potent, brain penetrant inhibitors of T. brucei NMT.
Subject(s)
Acyltransferases/antagonists & inhibitors , Aminopyridines/chemistry , Aminopyridines/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/enzymology , Trypanosomiasis, African/drug therapy , Acyltransferases/metabolism , Aminopyridines/chemical synthesis , Aminopyridines/pharmacokinetics , Animals , Brain/metabolism , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Humans , Mice , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/pharmacokinetics , Trypanosomiasis, African/metabolismABSTRACT
BACKGROUND: Protein kinases have been shown to be key drug targets, especially in the area of oncology. It is of interest to explore the possibilities of protein kinases as a potential target class in Plasmodium spp., the causative agents of malaria. However, protein kinase biology in malaria is still being investigated. Therefore, rather than assaying against individual protein kinases, a library of 4731 compounds with protein kinase inhibitor-like scaffolds was screened against the causative parasite, Plasmodium falciparum. This approach is more holistic and considers the whole kinome, making it possible to identify compounds that inhibit more than one P. falciparum protein kinase, or indeed other malaria targets. RESULTS: As a result of this screen, 9 active compound series were identified; further validation was carried out on 4 of these series, with 3 being progressed into hits to lead chemistry. The detailed evaluation of one of these series is described. DISCUSSION: This screening approach proved to be an effective way to identify series for further optimisation against malaria. Compound optimisation was carried out in the absence of knowledge of the molecular target. Some of the series had to be halted for various reasons. Mode of action studies to find the molecular target may be useful when problems prevent further chemical optimisation. CONCLUSIONS: Progressible series were identified through phenotypic screening of a relatively small focused kinase scaffold chemical library.
Subject(s)
Antimalarials/pharmacology , Plasmodium falciparum/drug effects , Protein Kinase Inhibitors/pharmacology , Drug Evaluation, PreclinicalABSTRACT
In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.
Subject(s)
Amines/pharmacology , DNA Replication/drug effects , DNA/metabolism , Origin Recognition Complex/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Amines/chemistry , Amines/metabolism , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chromatin/chemistry , Chromatin/metabolism , Humans , Minichromosome Maintenance Proteins/chemistry , Minichromosome Maintenance Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Origin Recognition Complex/antagonists & inhibitors , Quinolines/pharmacology , Replication Origin/genetics , Thiazoles/pharmacology , Xenopus , Xenopus Proteins/metabolismABSTRACT
The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.
Subject(s)
Antimalarials/pharmacology , Drug Discovery , Malaria/drug therapy , Plasmodium falciparum/drug effects , Quinolines/pharmacology , Animals , Antimalarials/chemical synthesis , Antimalarials/chemistry , Disease Models, Animal , Mice , Molecular Structure , Quinolines/chemical synthesis , Quinolines/chemistry , Structure-Activity RelationshipABSTRACT
In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting.
Subject(s)
Antimalarials/chemistry , Antimalarials/therapeutic use , Malaria/drug therapy , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Animals , Antimalarials/pharmacokinetics , Antimalarials/pharmacology , Humans , Malaria/parasitology , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Mice, SCID , Parasitemia/drug therapy , Parasitemia/parasitology , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacologyABSTRACT
The enzyme N-myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high-throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X-ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X-ray crystallography/structure-based design approach, the benzomorpholinone series was further optimised, increasing activity against T.â brucei NMT by >1000-fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T.â brucei.
Subject(s)
Acyltransferases/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Trypanosoma brucei brucei/enzymology , Acyltransferases/metabolism , Binding Sites/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Trypanosoma brucei brucei/drug effectsABSTRACT
A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5â µm. Screening of these hits in a T.â b.â brucei proliferation assay highlighted three compounds with a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold-hopping exercise led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaffold, which retained potency. A number of examples were assessed in a T.â b.â brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H-imidazo[4,5-b]pyrazin-2(3H)-one series were found to be either static or growth-slowing and not cidal. Compounds with the 1H-pyrazolo[3,4-b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.
Subject(s)
Drug Discovery , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Small Molecule Libraries/pharmacology , Trypanosoma brucei rhodesiense/drug effects , Cell Line , Dose-Response Relationship, Drug , Humans , Molecular Structure , Parasitic Sensitivity Tests , Phenotype , Protein Kinase Inhibitors/chemistry , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Trypanosoma brucei rhodesiense/growth & developmentABSTRACT
There is an urgent need for new, brain penetrant small molecules that target the central nervous system second stage of human African trypanosomiasis (HAT). We report that a series of novel indoline-2-carboxamides have been identified as inhibitors of Trypanosoma brucei from screening of a focused protease library against Trypanosoma brucei brucei in culture. We describe the optimization and characterization of this series. Potent antiproliferative activity was observed. The series demonstrated excellent pharmacokinetic properties, full cures in a stage 1 mouse model of HAT, and a partial cure in a stage 2 mouse model of HAT. Lack of tolerability prevented delivery of a fully curative regimen in the stage 2 mouse model and thus further progress of this series.
Subject(s)
Brain/drug effects , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosomiasis, African/drug therapy , Animals , Chemistry Techniques, Synthetic , Disease Models, Animal , Drug Discovery , Drug Evaluation, Preclinical/methods , Female , Indoles/chemistry , Mice, Inbred Strains , Stereoisomerism , Structure-Activity Relationship , Trypanocidal Agents/pharmacokinetics , Trypanosoma brucei brucei/growth & development , Trypanosomiasis, African/parasitologyABSTRACT
There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.
Subject(s)
Antimalarials/pharmacology , Gene Expression Regulation/drug effects , Malaria/parasitology , Plasmodium/drug effects , Plasmodium/metabolism , Protein Biosynthesis/drug effects , Quinolines/pharmacology , Animals , Antimalarials/administration & dosage , Antimalarials/adverse effects , Antimalarials/pharmacokinetics , Drug Discovery , Female , Life Cycle Stages/drug effects , Liver/drug effects , Liver/parasitology , Malaria/drug therapy , Male , Models, Molecular , Peptide Elongation Factor 2/antagonists & inhibitors , Peptide Elongation Factor 2/metabolism , Plasmodium/genetics , Plasmodium/growth & development , Plasmodium berghei/drug effects , Plasmodium berghei/physiology , Plasmodium falciparum/drug effects , Plasmodium falciparum/metabolism , Plasmodium vivax/drug effects , Plasmodium vivax/metabolism , Quinolines/administration & dosage , Quinolines/chemistry , Quinolines/pharmacokineticsABSTRACT
Trypanosoma brucei N-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.
Subject(s)
Acyltransferases/antagonists & inhibitors , Pyrazoles/chemical synthesis , Sulfonamides/chemical synthesis , Trypanocidal Agents/chemical synthesis , Trypanosomiasis, African/drug therapy , Aminopyridines/chemistry , Animals , Blood-Brain Barrier/drug effects , Central Nervous System/drug effects , Female , Humans , Inhibitory Concentration 50 , Mice , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effectsABSTRACT
Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3ß enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture.
Subject(s)
Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Trypanosomiasis, African/drug therapy , Adenosine Triphosphate/metabolism , Binding Sites , Chemistry Techniques, Synthetic , Cyclin-Dependent Kinase 2/chemistry , Cyclin-Dependent Kinase 2/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Humans , Models, Molecular , Protein Conformation , Pyrazoles/chemistry , Pyrazoles/therapeutic use , Substrate SpecificitySubject(s)
Academies and Institutes/organization & administration , Contract Services/organization & administration , Drug Discovery/methods , Technology, Pharmaceutical/organization & administration , Academies and Institutes/economics , Academies and Institutes/standards , Capacity Building/organization & administration , Capacity Building/standards , Contract Services/economics , Contract Services/methods , Contract Services/standards , Drug Discovery/economics , Drug Discovery/organization & administration , Interdisciplinary Communication , Technology, Pharmaceutical/economics , Technology, Pharmaceutical/methodsABSTRACT
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.
Subject(s)
Nucleotidyltransferases/antagonists & inhibitors , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/enzymology , Allosteric Regulation/drug effects , Catalytic Domain , Humans , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Protein Conformation , Trypanosoma brucei brucei/growth & development , Trypanosomiasis, African/drug therapy , Trypanosomiasis, African/parasitology , Uridine Diphosphate N-Acetylglucosamine/metabolismABSTRACT
Human African trypanosomiasis (HAT) is a life-threatening disease with approximately 30 000-40 000 new cases each year. Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is required for parasite growth and survival. Herein we report a screen of a focused kinase library against T.â brucei GSK3. From this we identified a series of several highly ligand-efficient TbGSK3 inhibitors. Following the hit validation process, we optimised a series of diaminothiazoles, identifying low-nanomolar inhibitors of TbGSK3 that are potent inâ vitro inhibitors of T.â brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.
Subject(s)
Drug Discovery , Glycogen Synthase Kinase 3/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma brucei brucei/drug effects , Trypanosoma brucei brucei/enzymology , Cell Line , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Glycogen Synthase Kinase 3/metabolism , Humans , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/chemistryABSTRACT
Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.
Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania donovani/drug effects , Leishmaniasis, Visceral/drug therapy , Parasitic Sensitivity Tests , Axenic Culture , Cell Line , Drug Discovery , High-Throughput Screening Assays , Humans , Macrophages/parasitologyABSTRACT
The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness, and there is an urgent unmet need for improved treatments. Parasite protein kinases are attractive drug targets, provided that the host and parasite kinomes are sufficiently divergent to allow specific inhibition to be achieved. Current drug discovery efforts are hampered by the fact that comprehensive assay panels for parasite targets have not yet been developed. Here, we employ a kinase-focused chemoproteomics strategy that enables the simultaneous profiling of kinase inhibitor potencies against more than 50 endogenously expressed T. brucei kinases in parasite cell extracts. The data reveal that T. brucei kinases are sensitive to typical kinase inhibitors with nanomolar potency and demonstrate the potential for the development of species-specific inhibitors.