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According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Chemical modulation of the flavonol 2-(benzo[d][1,3]dioxol-5-yl)-chromen-4-one (1), a promising anti-Trypanosomatid agent previously identified, was evaluated through a phenotypic screening approach. Herein, we have performed structure–activity relationship studies around hit compound 1. The pivaloyl derivative (13) showed significant anti-T. brucei activity (EC50 = 1.1 μM) together with a selectivity index higher than 92. The early in vitro ADME-tox properties (cytotoxicity, mitochondrial toxicity, cytochrome P450 and hERG inhibition) were determined for compound 1 and its derivatives, and these led to the identification of some liabilities. The 1,3-benzodioxole moiety in the presented compounds confers better in vivo pharmacokinetic properties than those of classical flavonols. Further studies using different delivery systems could lead to an increase of compound blood levels.

Aberrant activation of caspase-6 (C6) in the absence of other hallmarks of apoptosis has been demonstrated in cells and tissues from patients with Huntington disease (HD) and animal models. C6 activity correlates with disease progression in patients with HD and the cleavage of mutant huntingtin (mHTT) protein is thought to strongly contribute to disease pathogenesis. Here we show that the mHTT1-586 fragment generated by C6 cleavage interacts with the zymogen form of the enzyme, stabilizing a conformation that contains an active site and is prone to full activation. This shift toward enhanced activity can be prevented by a small-molecule inhibitor that blocks the interaction between C6 and mHTT1-586. Molecular docking studies suggest that the inhibitor binds an allosteric site in the C6 zymogen. The interaction of mHTT1-586 with C6 may therefore promote a self-reinforcing, feedforward cycle of C6 zymogen activation and mHTT cleavage driving HD pathogenesis.

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 mM for Leishmania infantum, 3.4 mM for L. donovani, 6.7 mM for L. major), Trypanosoma cruzi (EC50 7.5 mM) and T. brucei (EC50 0.8 mM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6] uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>10(7)M(-1)) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters (k(cat) = 0.12 s(-1) and K-M = 24 mu M) and inhibition constants of the two ODC inhibitors alpha-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)-compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni(2+)-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

Protein phosphatases (PP) are interesting drug targets. However, their ubiquitous presence and involvement in different, partially opposing signal pathways suggest that specificity may be achieved rather by targeting their interaction with subunits determining substrate specificity than the enzyme itself. An interesting subunit is phosphatase inhibitor-1 (I-1), which, in its protein kinase A-phosphorylated form (I-1(P)), inhibits the catalytic subunit of type 1 phosphatase (PP1c). In the current study, we established a colorimetric and a fluorescence-based assay system for the identification of compounds interfering with the inhibitory effect of I-1(P) on PP1c. The fluorescence assay exhibited 500-fold higher sensitivity toward PP1c. A nine-residue peptide containing the PP1c-binding motif (RVxF) of I-1 stimulated PP1c activity in the presence of I-1(P) (EC50 27 µM and 2.3 µM in the colorimetric and fluorescence assay, respectively). This suggests that the peptide interfered with the inhibitory effect of I-1(P) on PP1c and represents a proof-of-principle. The calculated Z' factor for PP1c (0.84) and the PP1c-I-1(P) complex (0.73) confirmed the suitability of the fluorescence assay for high-throughput screenings (HTS). By testing several thousand small molecules, we suggest the advantages of kinetic measurements over single-point measurements using the fluorescence-based assay in an HTS format. peerReviewed