Chemical Validation of Methionyl tRNA Synthetase (MetRS) as a Druggable Target in <i>Leishmania donovani</i>

Methionyl-tRNA synthetase (MetRS) has been chemically validated as a drug target in the kinetoplastid parasite Trypanosoma brucei. In the present study we investigate the validity of this target in the related trypanosomatid Leishmania donovani. Following development of a robust highthroughput compatible biochemical assay, a compound screen identified DDD806905 as a highly potent inhibitor of LdMetRS (Ki 18 nM). Crystallography revealed this compound binds to the methionine pocket of MetRS with enzymatic studies confirming DDD806905 displays competitive inhibition with respect to methionine and mixed inhibition with respect to ATP binding. DDD806905 showed activity, albeit with different levels of potency, in various Leishmania cell-based viability assays, with on-target activity observed in both Leishmania promastigote cell assays and a Leishmania tarentolae in vitro translation assay. Unfortunately this compound failed to show efficacy in an animal model of leishmaniasis. We investigated the potential causes for the discrepancies in activity observed in different Leishmania cell assays and the lack of efficacy in the animal model and found that high protein binding as well as sequestration of this dibasic compound into acidic compartments may play a role. Despite medicinal chemistry efforts to address the dibasic nature of DDD806905 and analogues, no progress could be achieved with the current chemical series. Although DDD806905 is not a developable anti-leishmanial compound, MetRS remains an attractive anti-leishmanial drug target

Pharmacological validation of N-myristoyltransferase as a drug target in <i>Leishmania donovani</i>

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ~30,000 deaths annually. Available treatments are inadequate and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1,600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesised with less basic centres. Although most of these compounds continued to suffer from relatively poor anti-leishmanial activity, our most potent inhibitor of LmNMT (DDD100097, Ki 0.34 nM), showed modest activity against L. donovani intracellular amastigotes (EC50 2.4 µM) and maintained a modest therapeutic window over the human enzyme. Two un-biased approaches, namely screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area

A Molecular Hybridization Approach for the Design of Potent, Highly Selective, and Brain-Penetrant N-Myristoyltransferase Inhibitors

Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood–brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease

Trisubstituted Pyrimidines as Efficacious and Fast-acting Antimalarials

In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting

Discovery of a Novel Class of Orally Active Trypanocidal N-Myristoyltransferase Inhibitors

N-Myristoyltransferase (NMT) represents a promising drug target for human African trypanosomiasis (HAT), which is caused by the parasitic protozoa Trypanosoma brucei. We report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound has good oral pharmacokinetics and cures rodent models of peripheral HAT infection. This compound provides an excellent tool for validation of T. brucei NMT as a drug target for HAT as well as a valuable lead for further optimization.</p

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis

The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis

There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 30 mg kg(-1) for 5 days resulted in sterile cures in a mouse model of VL. Treatment with lower doses revealed a U-shaped (hormetic) dose-response curve with greater parasite suppression at 1 mg kg(-1) than at 3 mg kg(-1) (5 or 10 day dosing). Dosing delamanid for 10 days confirmed the hormetic dose-response and improved the efficacy at all doses investigated. Mechanistic studies reveal that delamanid is rapidly metabolised by parasites via an enzyme, distinct from the nitroreductase that activates fexinidazole. Delamanid has the potential to be repurposed as a much-needed oral therapy for VL.</p

Discovery of a quinoline-4-carboxamide derivative with a novel mechanism of action, multistage antimalarial activity, and potent in vivo efficacy

The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi. Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.</p

Application of a mouse model humanized for cytochrome P450–mediated drug metabolism to predict drug-drug interactions between a peptide and small molecule drugs

Conventional preclinical in vitro approaches inaccurately predicted clinical trial outcomes of drug-drug interactions involving the peptide NN1177, a glucagon and glucagon-like peptide 1 receptor coagonist. To further study the mechanisms behind this discrepancy, we have exploited a mouse model (8HUM) humanized for the major cytochrome P450 (P450) enzymes involved in drug disposition in humans. We show that NN1177 administration to 8HUM mice suppressed hepatic in vivo expression of CYP3A4 (82% compared to vehicle) and CYP1A2 (58% compared to vehicle). This was consistent with in vitro sandwich culture hepatocyte data reported previously. However, reduction in CYP3A4 and CYP1A2 levels in vivo appeared to resolve over time, despite daily NN1177 administration. These findings suggest an adaptive response to the metabolic effects of NN1177. In vivo pharmacokinetic studies in 8HUM closely matched the findings observed in the clinical trial, because there was no relevant increase in the exposure of the CYP3A4 and CYP1A2 probe drugs. Furthermore, no suppression effects were observed when the mice had been pretreated with the inducing agents, St. John’s wort or phenobarbital, respectively, suggesting that the mechanism of P450 reduction does not involve the transcription factors constitutive androgen receptor or pregnane X receptor. These data highlight the complexities associated with therapeutic peptide drug-drug interactions and the remaining challenges for accurate predictions of P450 suppression and potential clinical implications. The humanized 8HUM model provides a promising and informative preclinical tool that can add high value during drug development by providing further insights into the effects on P450 expression, together with the subsequent impact of coadministered probe drugs in an in vivo model. Significance Statement: The current work describes the application of a humanized cytochrome P450 mouse model that provides further insight into the potential mechanisms and outperforms conventional in vitro approaches for preclinical predictions of peptide drug-drug interaction risk. The results showed no significant effects on the Cooperstown 5 + 1 cocktail, in line with clinical findings, and thereby represent an exciting model to further explore future therapeutic peptide projects during drug development.</p