Development of subunit selective proteasome substrates for Schistosoma species.

Schistosomiasis, or bilharzia, is a neglected tropical disease caused by Schistosoma spp. blood flukes that infects over 200 million people worldwide. Just one partially effective drug is available, and new drugs and drug targets would be welcome. The 20S proteasome is a validated drug target for many parasitic infections, including those caused by Plasmodium and Leishmania. We previously showed that anticancer proteasome inhibitors that act through the Schistosoma mansoni 20S proteasome (Sm20S) kill the parasite in vitro. To advance these initial findings, we employed Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to define the substrate cleavage specificities of the three catalytic ß subunits of purified Sm20S. The profiles in turn were used to design and synthesize subunit-specific optimized substrates that performed two to eight fold better than the equivalent substrates used to measure the activity of the constitutive human proteasome (c20S). These specific substrates also eliminated the need to purify Sm20S from parasite extracts - a single step enrichment was sufficient to accurately measure substrate hydrolysis and its inhibition with proteasome inhibitors. Finally, we show that the substrate and inhibition profiles for the 20S proteasome from the three medically important schistosome species are similar, suggesting that data arising from an inhibitor development campaign that focuses on Sm20S can be extrapolated to the other two targets with consequent time and cost savings.

Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies.

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

Structure-Activity Relationships, Tolerability and Efficacy of Microtubule-Active 1,2,4-Triazolo[1,5-a]pyrimidines as Potential Candidates to Treat Human African Trypanosomiasis.

Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT-active compounds hold promise as antitrypanosomal agents. MT-targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,ß-tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against cultured Trypanosoma brucei enabled a robust quantitative structure-activity relationship (QSAR) model and the prioritization of two congeners for in vivo pharmacokinetics (PK), tolerability and efficacy studies. Treatment of T. brucei-infected mice with tolerable doses of TPDs significantly decreased blood parasitemia within 24 h. Further, two once-weekly doses at 10 mg/kg of a candidate TPD significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS-active TPDs may provide alternative treatments for human African trypanosomiasis.

Protein-protein interactions: developing small-molecule inhibitors/stabilizers through covalent strategies.

The development of small-molecule inhibitors or stabilizers of selected protein-protein interactions (PPIs) of interest holds considerable promise for the development of research tools as well as candidate therapeutics. In this context, the covalent modification of selected residues within the target protein has emerged as a promising mechanism of action to obtain small-molecule modulators of PPIs with appropriate selectivity and duration of action. Different covalent labeling strategies are now available that can potentially allow for a rational, ground-up discovery and optimization of ligands as PPI inhibitors or stabilizers. This review article provides a synopsis of recent developments and applications of such tactics, with a particular focus on site-directed fragment tethering and proximity-enabled approaches.

Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

Structure-Activity Relationships, Tolerability and Efficacy of Microtubule-Active 1,2,4-Triazolo[1,5- a ]pyrimidines as Potential Candidates to Treat Human African Trypanosomiasis.

Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT- active compounds hold promise as antitrypanosomal agents. MT-targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,ß-tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against cultured Trypanosoma brucei enabled a robust quantitative structure-activity relationship (QSAR) model and the prioritization of two congeners for in vivo pharmacokinetics (PK), tolerability and efficacy studies. Treatment of T. brucei -infected mice with tolerable doses of TPDs 3 and 4 significantly decreased blood parasitemia within 24 h. Further, two once-weekly doses of 4 at 10 mg/kg significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS-active TPDs may provide alternative treatments for human African trypanosomiasis.

Activity of N-phenylbenzamide analogs against the neglected disease pathogen, Schistosoma mansoni.

For the Schistosoma mansoni flatworm pathogen, we report a structure-activity relationship of 25 derivatives of the N-phenylbenzamide compound, 1 (MMV687807), a Medicines for Malaria Venture compound for which bioactivity was originally identified in 2018. Synthesized compounds were cross-screened against the HEK 293 mammalian cells. Compounds 9 and 11 were identified as fast-acting schistosomicidal compounds whereby adult worm integrity was severely compromised within 1 h. Against HEK 293 mammalian cells, both compounds exhibited high CC50 values (9.8 ± 1.6 and 11.1 ± 0.2 µM respectively) which could translate to comfortable selectivity. When evaluated in a concentration-response format, compound 9 was active in the nanomolar range (EC50 = 80 nM), translating to a selectivity index of 123 over HEK 293 cells. The data encourage the further investigation of N-phenylbenzamides as antischistosomals.

Structure-activity relationship of dibenzylideneacetone analogs against the neglected disease pathogen, Trypanosoma brucei.

Trypanosoma brucei is a protozoan parasite that causes Human African Trypanosomiasis (HAT), a neglected tropical disease (NTD) that is endemic in 36 countries in sub-Saharan Africa. Only a handful drugs are available for treatment, and these have limitations, including toxicity and drug resistance. Using the natural product, curcumin, as a starting point, several curcuminoids and related analogs were evaluated against bloodstream forms of T. b. brucei. A particular subset of dibenzylideneacetone (DBA) compounds exhibited potent in vitro antitrypanosomal activity with sub-micromolar EC50 values. A structure-activity relationship study including 26 DBA analogs was initiated, and several compounds exhibited EC50 values as low as 200 nM. Cytotoxicity counter screens in HEK293 cells identified several compounds having selectivity indices above 10. These data suggest that DBAs offer starting points for a new small molecule therapy of HAT.

A Broad Spectrum Antiparasitic Activity of Organotin (IV) Derivatives and Its Untargeted Proteomic Profiling Using Leishmania donovani.

Metals have been used in medicine since ancient times for the treatment of different ailments with various elements such as iron, gold and arsenic. Metal complexes have also been reported to show antibiotic and antiparasitic activity. In this context, we tested the antiparasitic potential of 10 organotin (IV) derivatives from 4-(4-methoxyphenylamino)-4 oxobutanoic acid (MS26) against seven eukaryotic pathogens of medical importance: Leishmania donovani, Trypanosoma cruzi, Trypanosoma brucei, Entamoeba histolytica, Giardia lamblia, Naegleria fowleri and Schistosoma mansoni. Among the compounds with and without antiparasitic activity, compound MS26Et3 stood out with a 50% effective concentration (EC50) of 0.21 and 0.19 µM against promastigotes and intracellular amastigotes of L. donovani, respectively, 0.24 µM against intracellular amastigotes of T. cruzi, 0.09 µM against T. brucei, 1.4 µM against N. fowleri and impaired adult S. mansoni viability at 1.25 µM. In terms of host/pathogen selectivity, MS26Et3 demonstrated relatively mild cytotoxicity toward host cells with a 50% viability concentration of 4.87 µM against B10R cells (mouse monocyte cell line), 2.79 µM against C2C12 cells (mouse myoblast cell line) and 1.24 µM against HEK923 cells (human embryonic kidney cell line). The selectivity index supports this molecule as a therapeutic starting point for a broad spectrum antiparasitic alternative. Proteomic analysis of host cells infected with L. donovani after exposure to MS26Et3 showed a reduced expression of Rab7, which may affect the fusion of the endosome with the lysosome, and, consequently, impairing the differentiation of L. donovani to the amastigote form. Future studies to investigate the molecular target(s) and mechanism of action of MS26Et3 will support its chemical optimization.

Identification of Inhibitors of the Schistosoma mansoni VKR2 Kinase Domain.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms. Current treatment relies on just one partially effective drug, praziquantel (PZQ). Schistosoma mansoni Venus Kinase Receptors 1 and 2 (SmVKR1 and SmVKR2) are important for parasite growth and egg production, and are potential targets for combating schistosomiasis. VKRs consist of an extracellular Venus Flytrap Module (VFTM) linked via a transmembrane helix to a kinase domain. Here, we initiated a drug discovery effort to inhibit the activity of the SmVKR2 kinase domain (SmVKR2KD) by screening the GSK published kinase inhibitor set 2 (PKIS2). We identified several inhibitors, of which four were able to inhibit its enzymatic activity and induced phenotypic changes in ex vivo S. mansoni. Our crystal structure of the SmVKR2KD displays an active-like state that sheds light on the activation process of VKRs. Our data provide a basis for the further exploration of SmVKR2 as a possible drug target.

Anti-schistosomal activities of quinoxaline-containing compounds: From hit identification to lead optimisation.

Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold. Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against Schistosoma mansoni (1, EC50 = 4.59 µM on schistosomula). Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian (EC50 = 0.44 µM, 0.20 µM and 84.7 nM, on schistosomula, juvenile and adult worms, respectively) and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity (e.g. compound 30; EC50 = 2.59 nM on adult worms; selectivity index compared to the HepG2 cell line = 348). Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies.

A Machine Learning Strategy for Drug Discovery Identifies Anti-Schistosomal Small Molecules.

Schistosomiasis is a chronic and painful disease of poverty caused by the flatworm parasite Schistosoma. Drug discovery for antischistosomal compounds predominantly employs in vitro whole organism (phenotypic) screens against two developmental stages of Schistosoma mansoni, post-infective larvae (somules) and adults. We generated two rule books and associated scoring systems to normalize 3898 phenotypic data points to enable machine learning. The data were used to generate eight Bayesian machine learning models with the Assay Central software according to parasite's developmental stage and experimental time point (≤24, 48, 72, and >72 h). The models helped predict 56 active and nonactive compounds from commercial compound libraries for testing. When these were screened against S. mansoni in vitro, the prediction accuracy for active and inactives was 61% and 56% for somules and adults, respectively; also, hit rates were 48% and 34%, respectively, far exceeding the typical 1-2% hit rate for traditional high throughput screens.

Congeners Derived from Microtubule-Active Phenylpyrimidines Produce a Potent and Long-Lasting Paralysis of Schistosoma mansoni In Vitro.

Schistosomiasis is a parasitic disease that affects approximately 200 million people in developing countries. Current treatment relies on just one partially effective drug, and new drugs are needed. Tubulin and microtubules (MTs) are essential constituents of the cytoskeleton in all eukaryotic cells and considered potential drug targets to treat parasitic infections. The α- and ß-tubulin of Schistosoma mansoni have ∼96% and ∼91% sequence identity to their respective human tubulins, suggesting that compounds which bind mammalian tubulin may interfere with MT-mediated functions in the parasite. To explore the potential of different classes of tubulin-binding molecules as antischistosomal leads, we completed a series of in vitro whole-organism screens of a target-based compound library against S. mansoni adults and somules (postinfective larvae), and identified multiple biologically active compounds, among which phenylpyrimidines were the most promising. Further structure-activity relationship studies of these hits identified a series of thiophen-2-yl-pyrimidine congeners, which induce a potent and long-lasting paralysis of the parasite. Moreover, compared to the originating compounds, which showed cytotoxicity values in the low nanomolar range, these new derivatives were 1-4 orders of magnitude less cytotoxic and exhibited weak or undetectable activity against mammalian MTs in a cell-based assay of MT stabilization. Given their selective antischistosomal activity and relatively simple drug-like structures, these molecules hold promise as candidates for the development of new treatments for schistosomiasis.