Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Molecular dissection of Chagas induced cardiomyopathy reveals central disease associated and druggable signaling pathways.

Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.

Scaffold and Parasite Hopping: Discovery of New Protozoal Proliferation Inhibitors.

Utilizing a target repurposing and parasite-hopping approach, we tested a previously reported library of compounds that were active against Trypanosoma brucei, plus 31 new compounds, against a variety of protozoan parasites including Trypanosoma cruzi, Leishmania major, Leishmania donovani, and Plasmodium falciparum. This led to the discovery of several compounds with submicromolar activities and improved physicochemical properties that are early leads toward the development of chemotherapeutic agents against kinetoplastid diseases and malaria.

Design, Synthesis, and Biological Evaluation of New 1-(Aryl-1 H-pyrrolyl)(phenyl)methyl-1 H-imidazole Derivatives as Antiprotozoal Agents.

We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogues possess micromolar activities against Trypanosoma brucei rhodesiense and Leishmania donovani and nanomolar potency against Plasmodium falciparum. Most of the analogues displayed IC50 within the low nanomolar range against Trypanosoma cruzi, with very high selectivity toward the parasite. Discussion of structure-activity relationships and in vitro biological data for the new compounds are provided against a number of different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c, and 8b) was assessed by a target-based assay using recombinant T. cruzi CYP51. Bioavailability and efficacy of selected hits were assessed in a T. cruzi mouse model, where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal administration of 25 mg/kg/day dose for 4 consecutive days.

CYP51 is an essential drug target for the treatment of primary amoebic meningoencephalitis (PAM).

Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered "rare" (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug 'repurposing'-a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially 'druggable' target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

Binding mode and potency of N-indolyloxopyridinyl-4-aminopropanyl-based inhibitors targeting Trypanosoma cruzi CYP51.

Chagas disease is a chronic infection in humans caused by Trypanosoma cruzi and manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Limited therapeutic options to prevent and treat Chagas disease put 8 million people infected with T. cruzi worldwide at risk. CYP51, involved in the biosynthesis of the membrane sterol component in eukaryotes, is a promising drug target in T. cruzi. We report the structure-activity relationships (SAR) of an N-arylpiperazine series of N-indolyloxopyridinyl-4-aminopropanyl-based inhibitors designed to probe the impact of substituents in the terminal N-phenyl ring on binding mode, selectivity and potency. Depending on the substituents at C-4, two distinct ring binding modes, buried and solvent-exposed, have been observed by X-ray structure analysis (resolution of 1.95-2.48 Å). The 5-chloro-substituted analogs 9 and 10 with no substituent at C-4 demonstrated improved selectivity and potency, suppressing ≥ 99.8% parasitemia in mice when administered orally at 25 mg/kg, b.i.d., for 4 days.

4-Aminopyridyl-based CYP51 inhibitors as anti-Trypanosoma cruzi drug leads with improved pharmacokinetic profile and in vivo potency.

CYP51 is a P450 enzyme involved in the biosynthesis of the sterol components of eukaryotic cell membranes. CYP51 inhibitors have been developed to treat infections caused by fungi, and more recently the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. To specifically optimize drug candidates for T. cruzi CYP51 (TcCYP51), we explored the structure-activity relationship (SAR) of a N-indolyl-oxopyridinyl-4-aminopropanyl-based scaffold originally identified in a target-based screen. This scaffold evolved via medicinal chemistry to yield orally bioavailable leads with potent anti-T. cruzi activity in vivo. Using an animal model of infection with a transgenic T. cruzi Y luc strain expressing firefly luciferase, we prioritized the biaryl and N-arylpiperazine analogues by oral bioavailability and potency. The drug-target complexes for both scaffold variants were characterized by X-ray structure analysis. Optimization of both binding mode and pharmacokinetic properties of these compounds led to potent inhibitors against experimental T. cruzi infection.

R-Configuration of 4-Aminopyridyl-Based Inhibitors of CYP51 Confers Superior Efficacy Against Trypanosoma cruzi.

Sterol 14α-demethylase (CYP51) is an important therapeutic target for fungal and parasitic infections due to its key role in the biosynthesis of ergosterol, an essential component of the cell membranes of these pathogenic organisms. We report the development of potent and selective d-tryptophan-derived inhibitors of T. cruzi CYP51. Structural information obtained from the cocrystal structure of CYP51 and (R)-2, which is >1000-fold more potent than its enantiomer (S)-1, was used to guide design of additional analogues. The in vitro efficacy data presented here for (R)-2-(R)-8, together with preliminary in vitro pharmacokinetic data suggest that this new CYP51 inhibitor scaffold series has potential to deliver drug candidates for treatment of T. cruzi infections.

Rational development of 4-aminopyridyl-based inhibitors targeting Trypanosoma cruzi CYP51 as anti-chagas agents.

A new series of 4-aminopyridyl-based lead inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51) has been developed using structure-based drug design as well as structure-property relationship (SPR) analyses. The screening hit starting point, LP10 (KD ≤ 42 nM; EC50 = 0.65 µM), has been optimized to give the potential leads 14t, 27i, 27q, 27r, and 27t, which have low-nanomolar binding affinity to TcCYP51 and significant activity against T. cruzi amastigotes cultured in human myoblasts (EC50 = 14-18 nM for 27i and 27r). Many of the optimized compounds have improved microsome stability, and most are selective against human CYPs 1A2, 2D6, and 3A4 (<50% inhibition at 1 µM). A rationale for the improvement in microsome stability and selectivity of inhibitors against human metabolic CYP enzymes is presented. In addition, the binding mode of 14t with the Trypanosoma brucei CYP51 (TbCYP51) orthologue has been characterized by X-ray structure analysis.

Chemical-biological characterization of a cruzain inhibitor reveals a second target and a mammalian off-target.

Inhibition of the Trypanosoma cruzi cysteine protease cruzain has been proposed as a therapeutic approach for the treatment of Chagas' disease. Among the best-studied cruzain inhibitors to date is the vinylsulfone K777 (1), which has proven effective in animal models of Chagas' disease. Recent structure-activity studies aimed at addressing potential liabilities of 1 have now produced analogues such as N-[(2S)-1-[[(E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-3-(4-methylphenyl)-1-oxopropan-2-yl]pyridine-4-carboxamide (4), which is trypanocidal at ten-fold lower concentrations than for 1. We now find that the trypanocidal activity of 4 derives primarily from the inhibition of T. cruzi 14-α-demethylase (TcCYP51), a cytochrome P450 enzyme involved in the biosynthesis of ergosterol in the parasite. Compound 4 also inhibits mammalian CYP isoforms but is trypanocidal at concentrations below those required to significantly inhibit mammalian CYPs in vitro. A chemical-proteomics approach employing an activity-based probe derived from 1 was used to identify mammalian cathepsin B as a potentially important off-target of 1 and 4. Computational docking studies and the evaluation of truncated analogues of 4 reveal structural determinants for TcCYP51 binding, information that will be useful in further optimization of this new class of inhibitors.

Current understanding of the Trypanosoma cruzi-cardiomyocyte interaction.

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits multiple strategies to ensure its establishment and persistence in the host. Although this parasite has the ability to infect different organs, heart impairment is the most frequent clinical manifestation of the disease. Advances in knowledge of T. cruzi-cardiomyocyte interactions have contributed to a better understanding of the biological events involved in the pathogenesis of Chagas disease. This brief review focuses on the current understanding of molecules involved in T. cruzi-cardiomyocyte recognition, the mechanism of invasion, and on the effect of intracellular development of T. cruzi on the structural organization and molecular response of the target cell.

Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51.

BACKGROUND: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM. CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.

A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease.

A comparative study was performed between the trypanocidal efficacy of and associated immune response to benznidazole and posaconazole in a murine model of Chagas disease. Both drugs led to 100% survival, suppression of parasitaemia and reduction of specific anti-Trypanosoma cruzi antibodies following chronic infection. All posaconazole-treated animals had negative haemocultures at 54 days post infection, whilst 50% of those treated with benznidazole had positive results. Although both drugs were effective in reducing parasitism and inflammation in the heart, posaconazole-treated animals had plasma enzymatic levels of cardiac lesion that were indistinguishable from those of uninfected mice, whilst for benznidazole the enzyme levels were significantly higher than those of uninfected controls 31 days after the start of treatment. Posaconazole was more effective than benznidazole in controlling spleen enlargement and unspecific splenocyte proliferation in the early acute phase, but allowed higher levels of activation of CD4(+) and CD8(+) T-cells in the late acute phase when the adaptive immune response takes control of the infection. These results support the notion that posaconazole could be superior to benznidazole for the treatment of T. cruzi infection in humans.

Regulation of extracellular matrix expression and distribution in Trypanosoma cruzi-infected cardiomyocytes.

Alterations in the extracellular matrix have been observed in the cardiomyopathy of Chagas disease caused by Trypanosoma cruzi infection. However, the mechanism of extracellular matrix regulation in T. cruzi-infected cultured cardiomyocytes (CMs) is unclear. Using confocal laser microscopy, we demonstrated that treatment of these cultures with transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) leads to an enhancement of the fibronectin matrix only in uninfected CMs, while infected myocytes displayed low fibronectin expression. Digital image analysis also revealed low superposition of the fibronectin signal with parasite nests in cytokine treated and untreated cultures. Cytochalasin D treatment resulted in microfilament disarray that induced a disturbance in the fibronectin network of CMs, suggesting that cytoskeleton disruption caused by T. cruzi infection disorganizes the fibronectin matrix. Western blot analysis revealed a 2-fold increase in the fibronectin expression in CM cultures after cytokine treatment, whereas T. cruzi infection significantly reduced fibronectin levels in all conditions. In contrast, no change in the laminin expression was detected after cytokine treatment. Laminin distribution was altered in T. cruzi-infected CMs, with intense laminin labeling only at the cell periphery even after cytokine treatment. Our observations indicate that TGF-beta and TNF-alpha stimulates fibronectin expression only in uninfected cells of the T. cruzi-infected cultures, whereas the cells harboring the parasites display low or no fibronectin fibrils.

SB-431542, a transforming growth factor beta inhibitor, impairs Trypanosoma cruzi infection in cardiomyocytes and parasite cycle completion.

The antiinflammatory cytokine transforming growth factor beta (TGF-beta) plays an important role in Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-beta type I receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-beta pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion by T. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-beta signaling pathway in both T. cruzi infection and T. cruzi cell cycle completion. Our present data demonstrate that small inhibitors of the TGF-beta signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.

Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi.

Cytoadherence is an important step for the invasion of a mammalian host cell by Trypanosoma cruzi. Cell surface macromolecules are implicated in the T. cruzi-cardiomyocyte recognition process. Therefore, we investigated the role of cell surface proteoglycans during this invasion process and analyzed their expression after the parasite infected the target cells. Treatment of trypomastigote forms of T. cruzi with soluble heparan sulfate resulted in a significant inhibition in successful invasion, while chondroitin sulfate had no effect. Removal of sulfated glycoconjugates from the cardiomyocyte surface using glycosaminoglycan (GAG) lyases demonstrated the specific binding of the parasites to heparan sulfate proteoglycans. Infection levels were reduced by 42% whenthe host cells were previously treated with heparitinase II. No changes were detected in the expression of GAGs infected cardiomyocytes even after 96 h of infection. Our data demonstrate that heparan sulfate proteoglycans, but not chondroitin sulfate, mediate both attachment and invasion of cardiomyocytes by T. cruzi.