Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.     

Identification of a new class of nonpeptidic inhibitors of cruzain

Cruzain is the major cysteine protease of Trypanosoma cruzi, which is the causative agent of Chagas disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the substrate activity screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored beta-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group p K a, with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second-order inactivation constant of 147,000 s (-1) M (-1). This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas disease.     

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.     

Biogenesis of the reservosomes of Trypanosoma cruzi.

Reservosomes are endocytic compartments found in the posterior region of epimastigotes of Trypanosoma cruzi. In the differentiation from trypomastigotes to epimastigotes (reverse metacyclogenesis in vitro), one has the rare opportunity of following the biogenesis of an endocytic compartment. Metacyclic trypomastigotes incubated in LIT medium highly enriched with fetal calf serum differentiated directly to epimastigotes. In recently differentiated epimastigotes, acidic organelles were found in round compartments spread along the cell body, whereas in control epimastigotes they were found in reservosomes located in the posterior region. Ultrastructural analysis of intermediate forms showed that the cytostome and reservosomes appeared before differentiation to epimastigotes was completed. Many polymorphic reservosomes, with or without lipid inclusions, were observed from the anterior portion of the cell body, in close relationship with the Golgi complex, to the posterior region. Endocytic tracers were observed in the cytostome, flagellar pocket, vesicles, and newly formed reservosomes. Cruzipain, the main protease of T. cruzi, was localized in newly formed reservosomes and in vesicles budding from the trans-Golgi network that seem to fuse with reservosomes. Ingested gold-labeled albumin and cruzipain colocalized in recently formed reservosomes. Endocytosis and immunocytochemical analysis suggested that the endocytic and the secretory pathways may contribute to reservosome formation.     

Anti-virulence strategy against Brucella suis: synthesis, biological evaluation and molecular modeling of selective histidinol dehydrogenase inhibitors

In the facultative intracellular pathogen Brucella suis, histidinol dehydrogenase (HDH) activity, catalyzing the last step in histidine biosynthesis, is essential for intramacrophagic replication. The inhibition of this virulence factor by substituted benzylic ketones was a proof of concept that disarming bacteria leads to inhibition of intracellular bacterial growth in macrophage infection. This work describes the design, synthesis and evaluation of 19 new potential HDH inhibitors, using a combination of classical approaches and docking studies. The IC(50)-values of these inhibitors on HDH activity were in the nanomolar range, and several of them showed a 70-100% inhibition of Brucella growth in minimal medium. One selected compound yielded a strong inhibitory effect on intracellular replication of B. suis in human macrophages at concentrations as low as 5 μM, with an overall survival of intramacrophagic bacteria reduced by a factor 10(3). Docking studies with two inhibitors showed a good fitting in the catalytic pocket and also interaction with the second lipophilic pocket binding the cofactor NAD(+). Experimental data confirmed competition between inhibitors and NAD(+) at this site. Hence, these inhibitors can be considered as promising tools in the development of novel anti-virulence drugs.     

The new permeability pathways: targets and selective routes for the development of new antimalarial agents

The malaria parasite, Plasmodium falciparum, spends part of its complex life cycle within the red blood cells of a human host. During this time, the parasite alters the permeability of the red blood cell's plasma membrane to allow the uptake of nutrients, the removal of "waste" and volume and ion regulation of the infected cell. The increased permeability is due to the induction of new permeability pathways (NPP), which are obvious chemotherapeutic antimalarial targets and/or selective routes for drugs, which target the internal parasite. This review covers our present understanding of the NPP, the methods used to screen for putative inhibitors of the NPP, the current repertoire of NPP inhibitors and the problems that need to be addressed to realise the potential of the NPP as antimalarial targets. In addition, the review will cover the use of the NPP as specific drug delivery routes.     

ESR, electrochemical, molecular modeling and biological evaluation of 4-substituted and 1,4-disubstituted 7-nitroquinoxalin-2-ones as potential anti-Trypanosoma cruzi agents

Electrochemical and ESR studies were carried out in this work with the aim of characterizing the reduction mechanisms of 4-substituted and 1,4-disubstituted 7-nitroquinoxalin-2-ones by means of cyclic voltammetry in DMSO as aprotic solvent. Two reduction mechanisms were found for these compounds: the first, for compounds bearing a labile hydrogen by following a self-protonation mechanism (ECE steps), and the second, for compounds without labile hydrogen, based on a purely electrochemical reduction mechanism (typical of nitroheterocycles). The electrochemical results were corroborated using ESR spectroscopy allowing us to propose the hyperfine splitting pattern of the nitro-radical, which was later corroborated by the ESR simulation spectra. All these compounds were assayed as growth inhibitors against Trypanosoma cruzi: first, on the non-proliferative (and infective) form of the parasite (trypomastigote stage), and then, the ones that displayed activity, were assayed on the non-infective form (epimastigote stage). Thus, we found four new compounds highly active against T. cruzi. Finally, molecular modeling studies suggest the inhibition of the trypanothione reductase like one of the possible mechanisms involved in the trypanocidal action.     

Inactivation of Trypanosoma cruzi Trypomastigote Forms in Blood Components with a Psoralen and Ultraviolet A Light

Inactivation of the blood-borne parasite Trypanosoma cruzi by UVA and 4'-aminomethyl-4,5',8-trimethylpsor-alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo-reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form of T. cruzi , the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm². We note that while the infectivity of the parasite is eliminated at 4.2 J/cm^Z the trypomastigote motility continues for at least 16 h post-treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of ³H-AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborne T. cruzi , the causative agent of Chagas disease.     

INACTIVATION OF Trypanosoma cruzi TRYPOMASTIGOTE FORMS IN BLOOD COMPONENTS BY PHOTODYNAMIC TREATMENT WITH PHTHALOCYANINES

Abstract— -Three phthalocyanine dyes HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc 4), HOSiPc-OSi(CH₃)₂(CH₂)₃N+(CH₃)3I- (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS₄) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS₄ ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log₁₀) was found to occur with 2 μ M Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm², while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm². Even without illumination, Pc 4 at 2 μ M also killed about 3.7-4.1 log₁₀ of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.     

Steroidal Antimetabolites Protect Mice against Trypanosoma brucei

Trypanosoma brucei, the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT) that can equally target T. brucei. We found that CHT/ERGT inhibited cell growth in vitro, yielding EC₅₀ values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei, doubling their survival time following daily treatment for 8–10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.     

Trypanosomal dUTPases as potential targets for drug design

Parasites of the Trypanosomatidae family are responsible for diseases that afflict several million people worldwide. Currently there is an urgent need for new drugs against these diseases and an approach to drug discovery is the study of biochemical and structural properties of a potential target and the subsequent design of specific compounds. Trypanosomatid genes coding for enzymes which distinctively hydrolyze dUTP have been isolated by genetic complementation in Escherichia coli mutants defective in dUTPase activity. An analysis of these sequences from Leishmania major and Trypanosoma cruzi showed that no significant similarity could be established with the family of known dUTPases and that the five consensus motifs were absent. However, limited similarity was identified for three motifs present in an enzyme related in function the dCTPase-dUTPase from T phages and 35 percent identity with a putative dUTPase identified in the eubacteria Campylobacter jejuni. T. cruzi and L. major dUTPases were highly similar and catalyzed in a specific fashion the hydrolysis of dUTP. A detailed kinetic study of both enzymes revealed that dUDP is also an efficient substrate of the enzyme while other nucleotides are poorly hydrolyzed. The enzyme is essential for viability in Leishmania and is up-regulated by inhibitors of dTMP synthesis. Thus, a new family of dUTPases might exist in certain organisms that bear no sequence or structure similarity with eukaryotic enzymes accomplishing the same function and that may constitute potential drug targets for the development of specific inhibitors.     

Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

Development and characterization of an immobilized enzyme reactor (IMER) based on human glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

Immobilized enzyme reactors (IMERs) for on-line enzymatic studies are useful tool to select specific inhibitors and may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening to identify specific inhibitors. This technique has been shown to increase the stability of enzymes. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi and it has become a key target in the drug discovery program for Chagas' disease. Crystallographic studies have indicated that there are significant inter-species differences in GAPDH activity and sensitivity. For example the active sites of GAPDH in T. cruzi and humans differ by a substitution of ASP(210) (T. cruzi) by Leu(194) in human. Based on this information we initiated the study to develop optimal conditions for the covalent immobilization of the human GAPDH enzyme on a modified capillary support (400 mm x 0.10 mm). The chromatographic separation of NAD from NADH was achieved using a RP-Spherex-diol-OH (10 cm x 0.46 cm, 10 microm, 100 A) column. By using multidimensional HPLC chromatography system it was possible to investigate the activity and kinetic parameters of the GAPDH-IMER. The values obtained for D-GA3P and NAD were K(m)=3.5+/-0.2 mM and 0.75+/-0.04 mM, respectively, and were compared with values obtained with the free enzyme. The activity of the immobilized GAPDH has been preserved for over 120 days.     

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.     

Serum-stable RNA aptamers to an invariant surface domain of live African trypanosomes

African trypanosomes are extracellular blood parasites that cause sleeping sickness in humans and Nagana in cattle. The therapeutics used to control and treat these diseases are very ineffective and thus, the development of new drugs is urgently needed. We have previously suggested to use trypanosome-specific RNA aptamers as tools for the development of novel trypanocidal compounds. Here, we report the selection of a 2'-NH(2)-modified RNA aptamer that binds to live trypanosomes with an affinity of 70 +/- 15 nM. The aptamer adopts a stable G-quartet structure and has a half-life in human serum of > 30 h. RNA binding is restricted to the flagellar attachment zone, located between the cell body and the flagellum of the parasite. We demonstrate that antigen-tagged preparations of the aptamer can bind to live trypanosomes and that they can be used to re-direct immunoglobulins to the parasite surface.     

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi

Background: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas’ disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery.Results: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K_i’s between 0.5 and 17 μM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells.Conclusions: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas’ disease.     

Potent Inhibitors of Malarial Aspartic Proteases,the Plasmepsins,by Hydroformylation of Substituted 7‐Azanorbornenes

The increasing prevalence of multidrug‐resistant strains of the malarial parasite Plasmodium falciparum requires the urgent development of new therapeutic agents with novel modes of action. The vacuolar malarial aspartic proteases plasmepsin (PM) I, II, and IV are involved in hemoglobin degradation and play a central role in the growth and maturation of the parasite in the human host. We report the structure‐based design, synthesis, and in vitro evaluation of a new generation of PM inhibitors featuring a highly decorated 7‐azabicyclo[2.2.1]heptane core. While this protonated central core addresses the catalytic Asp dyad, three substituents bind to the flap, the S1/S3, and the S1′ pockets of the enzymes. A hydroformylation reaction is the key synthetic step for the introduction of the new vector reaching into the S1′ pocket. The configuration of the racemic ligands was confirmed by extensive NMR and X‐ray crystallographic analysis. In vitro biological assays revealed high potency of the new inhibitors against the three plasmepsins (IC₅₀ values down to 6 nM ) and good selectivity towards the closely related human cathepsins D and E. The occupancy of the S1′ pocket makes an essential contribution to the gain in binding affinity and selectivity, which is particularly large in the case of the PM IV enzyme. Designing non‐peptidic ligands for PM II is a valid route to generate compounds that inhibit the entire family of vacuolar plasmepsins.     

Identification of Mtb GlmU Uridyltransferase Domain Inhibitors by Ligand-Based and Structure-Based Drug Design Approaches

Targeting enzymes that play a role in the biosynthesis of the bacterial cell wall has long been a strategy for antibacterial discovery. In particular, the cell wall of Mycobacterium tuberculosis (Mtb) is a complex of three layers, one of which is Peptidoglycan, an essential component providing rigidity and strength. UDP-GlcNAc, a precursor for the synthesis of peptidoglycan, is formed by GlmU, a bi-functional enzyme. Inhibiting GlmU Uridyltransferase activity has been proven to be an effective anti-bacterial, but its similarity with human enzymes has been a deterrent to drug development. To develop Mtb selective hits, the Mtb GlmU substrate binding pocket was compared with structurally similar human enzymes to identify selectivity determining factors. Substrate binding pockets and conformational changes upon substrate binding were analyzed and MD simulations with substrates were performed to quantify crucial interactions to develop critical pharmacophore features. Thereafter, two strategies were applied to propose potent and selective bacterial GlmU Uridyltransferase domain inhibitors: (i) optimization of existing inhibitors, and (ii) identification by virtual screening. The binding modes of hits identified from virtual screening and ligand growing approaches were evaluated further for their ability to retain stable contacts within the pocket during 20 ns MD simulations. Hits that are predicted to be more potent than existing inhibitors and selective against human homologues could be of great interest for rejuvenating drug discovery efforts towards targeting the Mtb cell wall for antibacterial discovery.     

Design, synthesis and biological evaluation of potent azadipeptide nitrile inhibitors and activity-based probes as promising anti-Trypanosoma brucei agents

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small-molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain-like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile-containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target-based screening) and trypanocidal activity (i.e., whole-organism-based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity-based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease-relevant bloodstream form (BSF) and the insect-residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti-trypanosome agents, which possess better activities than existing drugs. The activity-based probes generated from this study could also serve as valuable tools for parasite-based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile-containing compounds as potential anti-parasitic agents.     

Systematic profiling of chemotherapeutic drug response to EGFR gatekeeper mutation in non-small cell lung cancer

The epidermal growth factor receptor (EGFR) targeted therapy has been established as a routine strategy for treating non-small cell lung cancer (NSCLC). However, the gatekeeper mutation T790M in EGFR active site can confer generic resistance to tyrosine kinase inhibitors (TKIs), largely limiting the clinical applications of chemotherapeutic drugs in NSCLC. Here, a combined method of computational analysis and growth inhibition assay was described to systematically investigate the molecular response profile of wild-type–sparing and mutant-resistant inhibitors to the EGFR T790M mutation. The profile is highly consistent with previous clinical observations; three first-line chemotherapeutic drugs Gefitinib, Erlotinib and Lapatinib are established with acquired resistance upon the mutation. In addition, it was found that the alkaloid compound K252a, a Staurosporine analog isolated from Nocardiopisis sp., can selectively target the EGFR T790M mutant over wild-type kinase (23-fold selectivity), suggesting that the compound is good lead candidate for development of T790M mutant-selective inhibitors. Structural analysis revealed that the mutation-resulting Met790 residue does not induce steric hindrance to the EGFR T790M–K252a complex system, while a number of hydrophobic forces, van der Waals contacts and S⋯π interactions are observed between the aromatic rings of K252a and the sulfhydryl group of Met790, contributing considerable stabilization energy to the system.