The parasite-mediated disease visceral leishmaniasis is prevalent in the tropics and causes 20,000–40,000 deaths across the globe each year1. The drug treatments currently used for this condition have substantial side effects, are difficult to administer and can result in the evolution of treatment-resistant parasite strains. Writing in Nature, Wyllie et al.2 present studies of a series of related drug-candidate molecules that are being developed for leishmaniasis treatment. They also identify the target of the most promising compound.
It is more than 100 years3 since drugs based on the chemical element antimony4 were first used to treat visceral leishmaniasis, also known as black fever or kala-azar. This sandfly-transmitted disease is caused by the protozoan parasites Leishmania donovani in the Old World and Leishmania infantum in both the Old World and the New World. Antimony-based drugs are still used today as part of a small range of treatments for the condition. In 2012, the World Health Organization reviewed5 the global impact of neglected tropical diseases in the developing world and identified the control of leishmaniasis worldwide and its elimination on the Indian subcontinent as priority targets. To achieve these goals, methods are needed to identify compounds with potent anti-parasitic activity that are suitable for safe and effective therapies.
Parasites from the Leishmania genus live and replicate inside a membrane-bound vacuole in macrophage cells of the immune system. Wyllie and colleagues studied compounds called pyrazolopyrimidines, which are effective against a related protozoan parasite, Trypanosoma brucei. The authors optimized the compounds by assessing their effect on in vitro infection of macrophages by Leishmania and by testing them using a mouse model of visceral leishmaniasis. They selected one named compound 7 as the best candidate for additional study because it had a good safety profile, high potency and suitable properties for development as an orally administered drug. However, the compound’s mode of action was unknown, so the authors sought to identify its molecular target in the parasite. Such target identification is important because it can aid the assessment of possible off-target effects in humans, as well as the likelihood of the emergence of drug resistance.
The authors used a biochemical approach to find proteins that bind to compound 7, and identified three enzymes of interest: CRK3, CRK6 and CRK12. These are similar to cyclin-dependent kinases (CDKs), protein kinases that need to bind a cyclin regulatory protein to enable their kinase activity6. When the authors isolated the enzymes, they were found to be associated with cyclin partners. CDKs regulate cell-cycle progression and are key anticancer drug targets6.
Wyllie and colleagues investigated parasites that had developed resistance to pyrazolopyrimidine compounds that was caused by exposing parasites to these molecules in the laboratory. The authors carried out whole-genome DNA sequencing to determine how this drug resistance had arisen. These results focused attention on CRK12, together with its associated cyclin protein CYC9, as the probable target of compound 7. This focus was supported by their finding that parasites that express both CRK12 and CYC9 at higher than usual levels have increased resistance to the effects of compound 7. Moreover, the authors identified a mutation in the CRK12 gene in drug-resistant parasites; when this mutation was introduced into wild-type parasites, they became resistant to compound 7 (Fig. 1). The authors’ computational modelling studies indicate that compound 7 binds CRK12 in the pocket where the molecule ATP usually binds.
Although CRK12 in complex with CYC9 seems to be the primary molecular target of compound 7, it is possible that other kinases in Leishmania could be inhibited by the molecule and contribute to its anti-parasitic activity. The range of protein kinases in Leishmania is different from that in humans, so this study provides an impetus to search for other ‘druggable’ protein kinases in the parasite7. Methods such as gene editing8 using the CRISPR–Cas9 technique have improved researchers’ ability to perform large-scale genetic validation of drug targets in Leishmania. However, a major bottleneck in the drug-discovery process for neglected tropical diseases is the identification of highly specific chemical probes that allow chemical validation — evidence that confirms the molecular target of a compound of interest.
The genetic and chemical validation of CRK12 as the target of pyrazolopyrimidines is a key advance because it opens further avenues of exploration for drug discovery. If the pyrazolopyrimidines ultimately fail to be suitable for clinical use, other compounds that inhibit CRK12 could be developed. There have been only a few drug-discovery efforts targeting enzymes in Leishmania9, mainly because not many targets have been genetically or chemically validated. In this instance, a target-based approach would require the production of CRK12 in complex with CYC9, and the development of an enzyme assay that would be suitable for high-throughput screening to test libraries of chemical compounds. Protein kinases are generally amenable to such approaches, but Wyllie and colleagues report that this has proved challenging so far for CRK12.
Further research should be carried out to investigate the regulation and function of the CRK12–CYC9 complex in Leishmania to determine whether modifications such as phosphorylation regulate the activity of the complex. One key question is why is this complex essential for the survival of Leishmania in its mammalian host? The authors found that compound 7 disrupts the parasite’s normal cell cycle, which is consistent with the known function of CDKs in cell division. However, the details of the molecular mechanisms at work here remain to be elucidated.
A study10 in 2016 identified the triazolopyrimidine molecule GNF6702 as having potent activity against Leishmania. It acts by inhibiting the cell’s proteasomal protein-degradation machinery. Thus, in the past few years, two promising compounds with known targets have emerged. Furthermore, collaborations between pharmaceutical companies, academic institutions and the non-profit Drugs for Neglected Diseases Initiative have identified an increasing number of candidate molecules for leishmaniasis treatment that could be orally administered; these might progress from preclinical studies to clinical trials (see go.nature.com/2lc3mgn).
Is it time to consider testing such chemicals in combination with each other? Combination therapy for visceral leishmaniasis is being evaluated for current drugs because this approach increases treatment efficacy, reduces treatment duration and limits or delays the emergence of drug resistance11. The use of lower concentrations of the compounds and shorter treatment times might help to avoid the emergence of difficult-to-treat Leishmania strains, such as those that have arisen after treatment with the drug miltefosine12. Wyllie and colleagues’ work might open the door for a new drug to be developed. Yet the attrition rate for drug candidates is high. More drug candidates therefore need to be identified to increase the chance that treatments for visceral leishmaniasis will make it to the clinic.
Nature 560, 171-172 (2018)