Treatment Of Human African Trypanosomiasis Research Articles

Transforming the chemotherapy of human African trypanosomiasis.

SUMMARYPrior to 2019, when the orally available drug fexinidazole began its clinical use, the treatment of human African trypanosomiasis (HAT) was complex and unsatisfactory for many reasons. Two sub-species of the Trypanosoma brucei parasite are responsible for HAT, namely the rhodesiense form found in East and Southern Africa and the gambiense form found in Central and West Africa. Diseases caused by both forms manifest in two stages: stage 1 before and stage 2 after central nervous system involvement. Prior to 2019, different drugs were required for each of the two parasite sub-species at each stage. Gambiense disease required pentamidine or nifurtimox-eflornithine combination therapy, while for rhodesiense disease, suramin or melarsoprol was given for stages 1 and 2, respectively. These drugs all suffered complications including protracted administration regimens involving multiple injections with drug-induced adverse effects common. Today, a single drug, fexinidazole, can be given orally in most cases for both diseases at either stage. Another compound, acoziborole, effective in both stages 1 and 2 gambiense disease with a single dosing is anticipated to become available within a few years. Moreover, the recent engagement of multilateral organizations in seeking other compounds that could be used in HAT therapy has also been successful, and a rich vein of new trypanocides has been discovered. Here, the clinical use, modes of action, and resistance risks for drugs used against HAT are discussed.

To Explore Potential Inhibitors Against Various Enzymatic Targets of Human African Trypanosomiasis.

Synthetic drugs currently prescribed for the treatment of Human African Trypanosomiasis (HAT) are non-specific, toxic, demand extended therapeutic regimes and are of varying efficacy. Along with the challenging demographic and socio-economic hurdles, the everincreasing risk of drug resistance is another major problem to be addressed. Cysteine protease, Heat shock proteins (HSP-90), Trypanothione reductase (TR), Farnesyl diphosphate synthase, Glucose-6-phosphate dehydrogenase, UP-4-galactose epimerase, and Cytidine triphosphate synthetase are potential enzymatic targets for the development of novel inhibitors against HAT which are the main focus of this review. The potential enzymatic targets of Trypanosoma brucei, especially small molecules like cysteine proteases and heat shock proteins are identified as major candidates for the sustenance of the parasite, their proliferation, infection, and spread of the disease. The development of new compounds to combat the disease by thorough ligand modification has been explored in the current review. Extracting these compounds and studying their efficacy, toxicity, and target mechanism extensively, this review has proposed a list of different compounds, including some synthetic and natural compounds along with multi-target inhibitors such as acoziborole, fexinidazole, etc. Potential inhibitors against these enzymatic targets of the T. brucei are important candidates for designing novel therapeutics against HAT. Multi-target inhibitors have also been identified as crucial molecules because of their potential advantage against the development of drug resistance.

Antigiardial and antiamebic activities of fexinidazole and its metabolites: new drug leads for giardiasis and amebiasis.

The intestinal parasites Giardia lamblia and Entamoeba histolytica are major causes of morbidity and mortality associated with diarrheal diseases. Metronidazole is the most common drug used to treat giardiasis and amebiasis. Despite its efficacy, treatment failures in giardiasis occur in up to 5%-40% of cases. Potential resistance of E. histolytica to metronidazole is an increasing concern. Therefore, it is critical to search for more effective drugs to treat giardiasis and amebiasis. We identified antigiardial and antiamebic activities of the rediscovered nitroimidazole compound, fexinidazole, and its sulfone and sulfoxide metabolites. Fexinidazole is equally active against E. histolytica and G. lamblia trophozoites, and both metabolites were 3- to 18-fold more active than the parent drug. Fexinidazole and its metabolites were also active against a metronidazole-resistant strain of G. lamblia. G. lamblia and E. histolytica cell extracts exhibited decreased residual nitroreductase activity when metabolites were used as substrates, indicating nitroreductase may be central to the mechanism of action of fexinidazole. In a cell invasion model, fexinidazole and its metabolites significantly reduced the invasiveness of E. histolytica trophozoites through basement membrane matrix. A q.d. oral dose of fexinidazole and its metabolites at 10 mg/kg for 3 days reduced G. lamblia infection significantly in mice compared to control. The newly discovered antigiardial and antiamebic activities of fexinidazole, combined with its FDA-approval and inclusion in the WHO Model List of Essential Medicines for the treatment of human African trypanosomiasis, offer decreased risk and a shortened development timeline toward clinical use of fexinidazole for treatment of giardiasis or amebiasis.

The evolving spectrum of human African trypanosomiasis.

Human African trypanosomiasis (HAT), or sleeping sickness, continues to be a major threat to human health in 36 countries throughout sub-Saharan Africa with up to 60 million people at risk. Over the last decade, there have been several advances in this area, some of which are discussed in this overview. Due to the concerted efforts of several bodies, including better identification and treatment of cases and improved tsetse fly vector control, the number of cases of HAT has declined dramatically. The clinical heterogeneity of HAT has also been increasingly recognized, and the disease, while usually fatal if untreated or inadequately treated, does not always have a uniformly fatal outcome. Improved methods of HAT diagnosis have now been developed including rapid diagnostic tests. Novel drug treatment of HAT has also been developed, notably nifurtimox-eflornithine combination therapy (NECT) for late-stage Trypanosoma brucei gambiense, oral fexinidazole for early and the early component of the late-stage of T.b. gambiense, and the new oral compounds of the oxaborole group, which have shown considerable promise in field trials. Advances in HAT neuropathogenesis have been steady, though largely incremental, with a particular focus on the role of the blood-brain barrier in parasite entry into the central nervous system and the relevant importance of both innate and adaptive immunity. While the World Health Organization goal of elimination of HAT as a public health problem by 2020 has probably been achieved, it remains to be seen whether the second more ambitious goal of interruption of transmission of HAT by 2030 will be attained.

Tackling Sleeping Sickness: Current and Promising Therapeutics and Treatment Strategies.

Human African trypanosomiasis is a neglected tropical disease caused by the extracellular protozoan parasite Trypanosoma brucei, and targeted for eradication by 2030. The COVID-19 pandemic contributed to the lengthening of the proposed time frame for eliminating human African trypanosomiasis as control programs were interrupted. Armed with extensive antigenic variation and the depletion of the B cell population during an infectious cycle, attempts to develop a vaccine have remained unachievable. With the absence of a vaccine, control of the disease has relied heavily on intensive screening measures and the use of drugs. The chemotherapeutics previously available for disease management were plagued by issues such as toxicity, resistance, and difficulty in administration. The approval of the latest and first oral drug, fexinidazole, is a major chemotherapeutic achievement for the treatment of human African trypanosomiasis in the past few decades. Timely and accurate diagnosis is essential for effective treatment, while poor compliance and resistance remain outstanding challenges. Drug discovery is on-going, and herein we review the recent advances in anti-trypanosomal drug discovery, including novel potential drug targets. The numerous challenges associated with disease eradication will also be addressed.

Current Prospects of Saponins as Promising Anti-Trypanosoma brucei Compounds: Insight into the Mechanisms of Action.

Human African trypanosomiasis (HAT) is a parasitic infection that may lead to death if left untreated. This disease is caused by a protozoan parasite of the genus Trypanosoma and is transmitted to humans through tsetse fly bites. The disease is widespread across Sub-Saharan Africa, with 70% of cases in recent reports in the Democratic Republic of the Congo and an average of less than 1000 cases are declared annually. Since there is no appropriate treatment for HAT, steroidal and triterpenoid saponins have been reported to be effective in in vitro studies and might serve as scaffolds for the discovery of new treatments against this disease. The present study aimed to summarize up-to-date information on the anti-Trypanosoma brucei activity of steroidal and triterpenoid saponins. The mechanisms of action of in vitro bioactive compounds were also discussed. Information on the anti-Trypanosoma brucei activity of plant saponins was obtained from published articles, dissertations, theses, and textbooks through a variety of libraries and electronic databases. There has been incredible progress in the identification of steroidal and triterpenoid saponins with pronounced in vitro activity against Trypanosoma brucei. Indeed, more than forty saponins were identified as having anti-T. brucei effect with activity ranging from moderate to highly active. The mechanisms of action of most of these saponins included DNA damage, cell cycle arrest, induction of apoptosis through downregulation of bcl-2 and MDM2, and upregulation of Bax and Bak, among others. Referring to in vitro studies, plant saponins have shown anti-Trypanosoma brucei activity; however, more cytotoxic and in vivo studies and detailed mechanisms of action of the bioactive saponins should be further considered.

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,β-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.

Development of a 2,4-Diaminothiazole Series for the Treatment of Human African Trypanosomiasis Highlights the Importance of Static-Cidal Screening of Analogues.

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.

Sleeping sickness: time for dreaming.

For a long time, the treatment of human African trypanosomiasis (HAT) represented for clinicians a journey through time, to the sources of colonial tropical medicine. Until 2018, the drugs available (apart from eflornithine), were commercialised during the first half of the 20th century.1 The pharmaceutical industry had no financial motivation to seek new products for a disease with a market representing a few tens of thousands of cases per year, living in rural and impoverished regions of sub-Saharan Africa.

Delivery of melarsoprol using folate-targeted PEGylated cyclodextrin-based nanoparticles for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, and has become one of the most lethal malignancies in the world. Although chemotherapy remains a cornerstone of cancer therapy, the number of chemotherapeutic drugs approved for HCC is low, and emerging therapeutics are needed. Melarsoprol (MEL) is an arsenic-containing drug, and has been applied in the treatment of human African trypanosomiasis at the late stage. In this study, the potential of MEL for HCC therapy was investigated for the first time using in vitro and in vivo experimental approaches. A folate-targeted polyethylene glycol-modified amphiphilic cyclodextrin nanoparticle was developed for safe, efficient and specific delivery of MEL. Consequently, the targeted nanoformulation achieved cell-specific uptake, cytotoxicity, apoptosis and migration inhibition in HCC cells. Furthermore, the targeted nanoformulation significantly prolonged the survival of mice with orthotopic tumor, without causing toxic signs. This study indicates the potential of the targeted nanoformulation as an emerging chemotherapy option for treating HCC.

Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030.

Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei. The subspecies T. b. gambiense is mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting for roughly 95% of all HAT cases. Trypanosoma b. rhodesiense results in more acute zoonotic infections in East-Africa. Because HAT has a two-stage pathogenesis, treatment depends on clinical assessment of patients and the determination whether or not parasites have crossed the blood brain barrier. Today, ultimate confirmation of parasitemia is still done by microscopy analysis. However, the introduction of diagnostic lateral flow devices has been a major contributor to the recent dramatic drop in T. b. gambiense HAT. Other techniques such as loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA)-based tests have been published but are still not widely used in the field. Most recently, CRISPR-Cas technology has been proposed to improve the intrinsic diagnostic characteristics of molecular approaches. This will become crucial in the near future, as preventing the resurgence of HAT will be a priority and will require tools with extreme high positive and negative predicted values, as well as excellent sensitivity and specificity. As for treatment, pentamidine and suramin have historically been the drugs of choice for the treatment of blood-stage gambiense-HAT and rhodesiense-HAT, respectively. For treatment of second-stage infections, drugs that pass the blood brain barrier are needed, and melarsoprol has been effectively used for both forms of HAT in the past. However, due to the high occurrence of post-treatment encephalopathy, the drug is not recommended for use in T. b. gambiense HAT. Here, a combination therapy of eflornithine and nifurtimox (NECT) has been the choice of treatment since 2009. As this treatment requires IV perfusion of eflornithine, efforts were launched in 2003 by the drugs for neglected disease initiative (DNDi) to find an oral-only therapy solution, suitable for rural sub-Saharan Africa treatment conditions. In 2019 this resulted in the introduction of fexinidazole, with a treatment regimen suitable for both the blood-stage and non-severe second-stage T. b. gambiense infections. Experimental treatment of T. b. rhodesiense HAT has now been initiated as well.

Fexinidazole for Human African Trypanosomiasis, the Fruit of a Successful Public-Private Partnership.

After 100 years of chemotherapy with impractical and toxic drugs, an oral cure for human African trypanosomiasis (HAT) is available: Fexinidazole. In this case, we review the history of drug discovery for HAT with special emphasis on the discovery, pre-clinical development, and operational challenges of the clinical trials of fexinidazole. The screening of the Drugs for Neglected Diseases initiative (DNDi) HAT-library by the Swiss TPH had singled out fexinidazole, originally developed by Hoechst (now Sanofi), as the most promising of a series of over 800 nitroimidazoles and related molecules. In cell culture, fexinidazole has an IC50 of around 1 µM against Trypanosoma brucei and is more than 100-fold less toxic to mammalian cells. In the mouse model, fexinidazole cures both the first, haemolymphatic, and the second, meningoencephalitic stage of the infection, the latter at 100 mg/kg twice daily for 5 days. In patients, the clinical trials managed by DNDi and supported by Swiss TPH mainly conducted in the Democratic Republic of the Congo demonstrated that oral fexinidazole is safe and effective for use against first- and early second-stage sleeping sickness. Based on the positive opinion issued by the European Medicines Agency in 2018, the WHO has released new interim guidelines for the treatment of HAT including fexinidazole as the new therapy for first-stage and non-severe second-stage sleeping sickness caused by Trypanosoma brucei gambiense (gHAT). This greatly facilitates the diagnosis and treatment algorithm for gHAT, increasing the attainable coverage and paving the way towards the envisaged goal of zero transmission by 2030.

Analytical assays to evaluate enzymatic activity and screening of inhibitors for ornithine decarboxylase

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to produce putrescine, the first step in the metabolism of polyamines (putrescine, spermidine, and spermine), which are essential growth factors in eukaryotic cells. ODC is active as a homodimer and depends on pyridoxal 5′-phosphate (PLP) as a cofactor. An increase in the concentration of polyamines has been associated with carcinogenesis. Therefore, there is much interest in identifying inhibitors of this pathway as potential chemotherapeutic and chemopreventive agents. The best-known inhibitor of mammalian ODC is α-difluoromethylornithine (DFMO), a highly selective compound that alkylates Cys-360 (a residue of the ODC active site). Although DFMO was initially developed for the treatment of cancer, the World Health Organization recommends its use in combination with nifurtimox for the treatment of human African trypanosomiasis. Considering the importance of ODC as a promising target for the treatment of various types of cancer and other infectious diseases, choosing the right method for screening potential inhibitors can help to accelerate the discovery of new drugs. Several methods for the determination of ODC activity are found in the literature. Among these, we can mention analysis with radioactive markers, colorimetric assays using auxiliary enzymes to detect CO2 or H2O2 release, chromatographic separations with putrescine derivatization, mass spectrometry, and spectroscopic techniques. In this review, the main analysis methods used will be described, highlighting their advantages and disadvantages, as well as identifying the most promising methods for high-throughput screening.

Preliminary Structure-Activity Relationship Study of the MMV Pathogen Box Compound MMV675968 (2,4-Diaminoquinazoline) Unveils Novel Inhibitors of Trypanosoma brucei brucei.

New drugs are urgently needed for the treatment of human African trypanosomiasis (HAT). In line with our quest for novel inhibitors of trypanosomes, a small library of analogs of the antitrypanosomal hit (MMV675968) available at MMV as solid materials was screened for antitrypanosomal activity. In silico exploration of two potent antitrypanosomal structural analogs (7-MMV1578647 and 10-MMV1578445) as inhibitors of dihydrofolate reductase (DHFR) was achieved, together with elucidation of other antitrypanosomal modes of action. In addition, they were assessed in vitro for tentative inhibition of DHFR in a crude trypanosome extract. Their ADMET properties were also predicted using dedicated software. Overall, the two diaminoquinazoline analogs displayed approximately 40-fold and 60-fold more potency and selectivity in vitro than the parent hit, respectively (MMV1578445 (10): IC50 = 0.045 µM, SI = 1737; MMV1578467 (7): IC50 = 0.06 µM; SI = 412). Analogs 7 and 10 were also strong binders of the DHFR enzyme in silico, in all their accessible protonation states, and interacted with key DHFR ligand recognition residues Val32, Asp54, and Ile160. They also exhibited significant activity against trypanosome protein isolate. MMV1578445 (10) portrayed fast and irreversible trypanosome growth arrest between 4–72 h at IC99. Analogs 7 and 10 induced in vitro ferric iron reduction and DNA fragmentation or apoptosis induction, respectively. The two potent analogs endowed with predicted suitable physicochemical and ADMET properties are good candidates for further deciphering their potential as starting points for new drug development for HAT.

Communities' Perception, Knowledge, and Practices Related to Human African Trypanosomiasis in the Democratic Republic of Congo.

Background: The number of human African trypanosomiasis (HAT) cases in the Democratic Republic of Congo (DRC) has significantly reduced, thanks to more effective drugs and screening tools and regular mass screening. However, this potentially jeopardizes HAT control activities, especially community engagement. Methods: We used an ecological model framework to understand how various factors shape communities’ knowledge, perceptions, and behavior in this low endemicity context. Community members, frontline health providers, and policymakers were consulted using an ethnographic approach. Results: Communities in endemic areas are knowledgeable about causes, symptoms, and treatment of HAT, but this was more limited among young people. Few are aware of new HAT treatment or screening techniques. Participation in mass screening has declined due to many factors including fear and a lack of urgency, given the low numbers of cases. Delays in seeking medical care are due to confusion of HAT symptoms with those of other diseases and belief that HAT is caused by witchcraft. Conclusions: Community members see their role more in terms of vector control than participation in screening, referral, or accepting treatment. We propose recommendations for achieving sustainable community engagement, including development of an information and communication strategy and empowerment of communities to take greater ownership of HAT control activities.

Determination of Optimal Dosing Duration for Cotrimoxazole, Ciprofloxacin, Clarythromycin, And Tinidazole, Novel Oral Drugs for The Treatment of T.B.Gambiense Human African Trypanosomiasis: First-In-Human Studies

Background and Objectives: Fexinidazole is a 5-nitroim-idazole recently included in a clinical efficacy trial as an oral drug for the treatment of human African trypanoso-miasis (HAT). Preclinical studies showed it acts as a pharmacologically active prodrug with two key active metabolites: sulfoxide and sulfone (the most active metab- olite). The present studies aimed to determine other oral antibiotic regimen for the treatment of stage 2 sleeping sickness which could eventually also treat stage 1 patients, like Fexinidazole. Cotrimoxazole 960 mgs twice daily for two weeks, or one month if unsuccessful at two weeks of treatment; clarithromycin 250 mgs twice daily and tinidazole 500 mgs twice daily for one week or two weeks; and ciprofloxacin 500mgs twice daily for two weeks were administered. Methods: Cotrimozaxole, ciprofloxacin, clarithromycin, and tinidazole were assessed in 99 HAT positive children and adult male and female subjects of sub-Saharan African origin. Three initial first-in-human studies and two additional studies assessed a weekly, two weekly and monthly doses of the above drugs. Cotrimoxazole 960 mgs twice daily for two weeks, or one month if unsuccessful at two weeks of treatment; clarithromycin 250 mgs twice daily and tinidazole 500 mgs twice daily for one week or two weeks; and ciprofloxacin 500mgs twice daily for two weeks were administered. Results: The drugs were well-tolerated in the various doses. Conclusion: These studies show that cotrimoxazole, ciprofloxacin, clarithromycin and tinidazole can be safely assessed in patients as potential oral cure for both stages of Human African Trypanasomiasis.

Identification of Prazosin as a Potential Flagellum Attachment Zone 1(FAZ1) Inhibitor for the Treatment of Human African Trypanosomiasis.

Human African trypanosomiasis (HAT) remains a health threat to sub-Saharan Africa. The current treatments suffer from drug resistance and life-threatening side effects, making drug discovery for HAT still important. A high-throughput screening of the library of pharmaceutically active compounds identified prazosin, an α-adrenoceptor antagonist, that showed selective activity toward Trypanosoma brucei brucei. Furthermore, a series of prazosin analogues were examined, and overall, the new analogues had improved activity and selectivity. To elucidate the binding partner, a biotin-conjugated probe was synthesized, and a protein pulldown assay combined with a proteomic analysis identified the flagellum attachment zone 1 (FAZ1) filament as an interacting partner. Additionally, prazosin treatment resulted in dysfunction of the flagellum of trypanosome cells, which is indicative of a FAZ1 irregularity. We also examined the drug distribution by utilizing immunofluorescence with a designed fluorescent analogue that showed partial colocalization with FAZ1. With the activity of the prazosin analogues, a structure-activity relationship (SAR) was summarized for future lead optimization. Our findings provide a new group of FAZ1 inhibitors as novel antitrypanosomal agents.

Discovery of novel natural products as rhodesain inhibitors for human African trypanosomiasis using in silico techniques

Human African Trypanosomiasis (HAT) or sleeping sickness is caused by the Trypanosoma brucei rhodesiense, a subspecies of the Trypanosomatide family. The parasite is associated with high morbidity and mortality rate in both animals and humans, claimed to be more fatal than other vector-transmitted diseases such as malaria. The majority of existing medications are highly toxic, not effective in the late chronic phase of the disease, and require maximum dosages to fully eradicate the parasite. In this study, we used computational methods to find out natural products that inhibit the Rhodesain, a parasitic enzyme that plays an important role in the parasite's pathogenicity, multiplication, and ability to pass through the host's blood-brain barrier. A library of 270540 natural products from ZINC databases was processed by using e-pharmacophore hypnosis and screening procedures, molecular docking, ADMET processes, and MM-GBSA calculations. This led to the identification of 3 compounds (ZINC000096269390, ZINC000035485292, and ZINC000035485242) which were then subjected to molecular dynamics. The findings of this study showed excellent binding affinity and stability toward the Rhodesain and suggest they may be a hopeful treatment for HAT in the future if further clinical trials were performed. Communicated by Ramaswamy H. Sarma

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10–SPR19) as potential anti-HAT agents. The new molecules show Ki values in the low-micro/sub-micromolar range against rhodesain, coupled with k2nd values between 1314 and 6950 M−1 min−1. With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 µM towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agents

Exploring the Activity Profile of TbrPDEB1 and hPDE4 Inhibitors Using Free Energy Perturbation.

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by the parasite Trypanosoma brucei (T.b.). A validated target for the treatment of HAT is the parasitic T.b. cyclic nucleotide phosphodiesterase B1 (TbrPDEB1). Although nanomolar TbrPDEB1 inhibitors have been obtained, their activity against the off-target human PDE4 (hPDE4) is likely to lead to undesirable clinical side effects, such as nausea, emesis, and immune suppression. Thus, new and more selective TbrPDEB1 inhibitors are still needed. This retrospective study evaluated the free energy perturbation (FEP+) method to predict the affinity profiles of TbrPDEB1 inhibitors against hPDE4. We demonstrate that FEP+ can be used to accurately predict the activity profiles of these homologous proteins. Moreover, we show how FEP+ can overcome challenges like protein flexibility and high sequence conservation. This also implies that the method can be applied prospectively for the lead optimization campaigns to design new and more selective TbrPDEB1 inhibitors.