African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

Christel Brunschwig,a Nina Lawrence,a Dale Taylor,a Efrem Abay,a Mathew Njoroge,a Gregory S. Basarab,a Claire Le Manach,b Tanya Paquet,b Diego Gonzàlez Cabrera,b Aloysius T. Nchinda,b Carmen de Kock,a Lubbe Wiesner,c Paolo Denti,c David Waterson,d Benjamin Blasco,d Didier Leroy,d Michael J. Witty,d Cristina Donini,d James Duffy,d Sergio Wittlin,e,f Karen L. White,g Susan A. Charman,g Maria Belén Jiménez-Díaz,h Iñigo Angulo-Barturen,h Esperanza Herreros,h Francisco Javier Gamo,h Rosemary Rochford,i Dalu Mancama,j Theresa L. Coetzer,k Mariëtte E. van der Watt,l Janette Reader,l Lyn-Marie Birkholtz,l Kennan C. Marsh,m Suresh M. Solapure,n† John E. Burke,z Jacob A. McPhail,z Manu Vanaerschot,o David A. Fidock,o,p Paul V. Fish,q Peter Siegl,r Dennis A. Smith,s Grennady Wirjanata,t Rintis Noviyanti,u Ric N. Price,t,v Jutta Marfurt,t Kigbafori D. Silue,w Leslie J. Street,b Kelly Chibaleb,x,y

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943.

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC50 values of 0.15 nM against T. congolense and 1.3 nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10 mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

Since the appearance of resistance to the current front-line antimalarial treatments, ACTs (artemisinin combination therapies), the discovery of novel chemical entities to treat the disease is recognized as a major global health priority. From the GSK antimalarial set, we identified an aminoxadiazole with an antiparasitic profile comparable with artemisinin (1), with no cross-resistance in a resistant strains panel and a potential new mode of action. A medicinal chemistry program allowed delivery of compounds such as 19 with high solubility in aqueous media, an acceptable toxicological profile, and oral efficacy. Further evaluation of the lead compounds showed that in vivo genotoxic degradants might be generated. The compounds generated during this medicinal chemistry program and others from the GSK collection were used to build a pharmacophore model which could be used in the virtual screening of compound collections and potentially identify new chemotypes that could deliver the same antiparasitic profile.

Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2Rγnull mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.

Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure activity relationship studies around the central core of antimalarial imidazo-pyridazines were conducted. This study led to the identification of potent pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles. The lead compounds also proved to be very potent in the parasite liver and gametocyte stages, which makes them of high interest.

Based on the initial optimization of orally active antimalarial 2,4-diamino-thienopyrimidines and with the help of metabolite identification studies, a second generation of derivatives involving changes at the 2- and 4-positions of the thienopyrimidine core were synthesized. Improvements in the physiochemical properties resulted in the identification of 15a, 17a, 32, and 40 as lead molecules with improved in vivo exposure. Furthermore, analogue 40 exhibited excellent in vivo antimalarial activity when dosed orally at 50 mg/kg once daily for 4 days in the Plasmodium berghei mouse model, which is superior to the activity seen with previously reported compounds, and with a slightly improved hERG profile.

A novel series of 2,4-diaminothienopyrimidines with potential as antimalarials was identified from whole-cell high-throughput screening of a SoftFocus ion channel library. Synthesis and structure-activity relationship studies identified compounds with potent antiplasmodial activity and low in vitro cytotoxicity. Several of these analogues exhibited in vivo activity in the Plasmodium berghei mouse model when administered orally. However, inhibition of the hERG potassium channel was identified as a liability for this series.

A novel class of imidazopyridazines identified from whole cell screening of a SoftFocus kinase library was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant strain) and NF54 (sensitive strain). Structure activity relationship studies led to the identification of highly potent compounds against both strains. Compound 35 was highly active (IC50: K1 = 6.3 nM, NF54 = 7.3 nM) and comparable in potency to artesunate, and 35 exhibited 98% activity in the in vivo P. berghei mouse model (4-day test by Peters) at 4 x 50 mg/kg po. Compound 35 was also assessed against P. falciparum in the in vivo SCID mouse model where the efficacy was found to be more consistent with the in vitro activity. Furthermore, 35 displayed high (78%) rat oral bioavailability with good oral exposure and plasma half-life. Mice exposure at the same dose was 10-fold lower than in rat, suggesting lower oral absorption and/or higher metabolic clearance in mice.

Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites. Therefore, the discovery of new antimalarial medicines is focused on new drugs that act by novel mechanisms and are active against different P. falciparum development stages. Screening of a focused compound library for antiparasitic activity, lead to identification of a novel class of compounds with activity against P. falciparum, 2-aminopyridines. The selected hits were validated and subjected to a lead optimization program resulting in the pre-clinical candidate MMV390048. Here we report an unbiased chemoproteomics strategy for the identification of targets of MMV390048. An analogue of MMV390048 containing a primary amine function for immobilization in a permissive position was synthesized and covalently immobilized on sepharose beads. Affinity capturing of potential target proteins from a P. falciparum blood stage extract was performed in the absence and presence of an excess of MMV390048 in the extract to delineate target proteins for which capturing is competitively inhibited. All proteins captured by the beads were quantified by isotope tagging of tryptic peptides followed by LC-MS/MS. Notably MMV390048 competitively inhibited the binding of only a single protein, P. falciparum PI4 kinase, to the beads. However, the immobilization of a drug compound via a linker may not be compatible with the binding to all of its targets. Therefore, we also performed a capturing experiment with kinobeads, which represent a combination of immobilized promiscuous ATP competitive kinase inhibitors (Bantscheff et al., 2007). As in the previous experiment, PfPI4K was the only P. falciparum protein which exhibited a reduction of bead binding upon the addition of MMV390048 to the extract. Knowledge of the target will accelerate the drug discovery process.

Antiparasitic oral drugs have been associated to lipophilic molecules due to their intrinsic permeability. However, these kind of molecules are associated to numerous adverse effects, which have been extensively studied. Within the Tres Cantos Antimalarial Set (TCAMS) we have identified two small, soluble and simple hits that even presenting antiplasmodial activities in the range of 0.4-0.5 μM are able to show in vivo activity.

On the basis of our recent results on a novel series of imidazopyridazine-based antimalarials, we focused on identifying compounds with improved aqueous solubility and hERG profile while maintaining metabolic stability and in vitro potency. Toward this objective, 41 compounds were synthesized and evaluated for antiplasmodial activity against NF54 (sensitive) and K1 (multidrug resistant) strains of the malaria parasite Plasmodium falciparum and evaluated for both aqueous solubility and metabolic stability. Selected compounds were tested for in vitro hERG activity and in vivo efficacy in the P. berghei mouse model. Several compounds were identified with significantly improved aqueous solubility, good metabolic stability, and a clean hERG profile relative to a previous frontrunner lead compound. A sulfoxide-based imidazopyridazine analog 45, arising from a prodrug-like strategy, was completely curative in the Plasmodium berghei mouse model at 4 × 50 mg/kg po.

In the second part of this Miniperspectives series, we highlight our medicinal chemistry efforts involving progression of hits from whole cell high-throughput screening (HTS) of a Soft Focus kinase library against the malaria parasite Plasmodium falciparum. Successful SAR exploration in Hit-to-Lead and Lead Optimization efforts leading to the selection of a preclinical development candidate are demonstrated. Related efforts by researchers from Broad/Genzyme, Anacor, and GSK are briefly covered.