NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Hepatic spheroids used as an in vitro model to study malaria relapse.

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.

Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit.

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50<0.1µM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

PI4 Kinase Is a Prophylactic but Not Radical Curative Target in Plasmodium vivax-Type Malaria Parasites.

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo.

Pharmacokinetic-pharmacodynamic analysis of spiroindolone analogs and KAE609 in a murine malaria model.

Limited information is available on the pharmacokinetic (PK) and pharmacodynamic (PD) parameters driving the efficacy of antimalarial drugs. Our objective in this study was to determine dose-response relationships of a panel of related spiroindolone analogs and identify the PK-PD index that correlates best with the efficacy of KAE609, a selected class representative. The dose-response efficacy studies were conducted in the Plasmodium berghei murine malaria model, and the relationship between dose and efficacy (i.e., reduction in parasitemia) was examined. All spiroindolone analogs studied displayed a maximum reduction in parasitemia, with 90% effective dose (ED90) values ranging between 6 and 38 mg/kg of body weight. Further, dose fractionation studies were conducted for KAE609, and the relationship between PK-PD indices and efficacy was analyzed. The PK-PD indices were calculated using the in vitro potency against P. berghei (2× the 99% inhibitory concentration [IC99]) as a threshold (TRE). The percentage of the time in which KAE609 plasma concentrations remained at >2× the IC99 within 48 h (%T>TRE) and the area under the concentration-time curve from 0 to 48 h (AUC0-48)/TRE ratio correlated well with parasite reduction (R2=0.97 and 0.95, respectively) but less so for the maximum concentration of drug in serum (Cmax)/TRE ratio (R2=0.88). The present results suggest that for KAE609 and, supposedly, for its analogs, the dosing regimens covering a T>TRE of 100%, AUC0-48/TRE ratio of 587, and a Cmax/TRE ratio of 30 are likely to result in the maximum reduction in parasitemia in the P. berghei malaria mouse model. This information could be used to prioritize analogs within the same class of compounds and contribute to the design of efficacy studies, thereby facilitating early drug discovery and lead optimization programs.

Lead optimization of imidazopyrazines: a new class of antimalarial with activity on Plasmodium liver stages.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

Progressing the global antimalarial portfolio: finding drugs which target multiple Plasmodium life stages.

The number of novel antimalarial candidates entering preclinical development has seen an increase over the last several years. Most of these drug candidates were originally identified as hits coming from screening large chemical libraries specifically targeting the asexual blood stages of Plasmodium falciparum. Indeed, a large proportion of the current antimalarial arsenal has mainly targeted the asexual blood stage which is responsible for clinical symptoms of the disease. However, as part of the eradication agenda and to address resistance, any next-generation antimalarial should have additional activity on at least one other parasite life stage, i.e. gametocytocidal and/or tissue schizonticidal activity. We have applied this approach by screening compounds with intrinsic activity on asexual blood stages in assays against sexual and liver stages and identified two new antimalarial chemotypes with activity on multiple parasite life stages. This strategy can be expanded to identify other chemical classes of molecules with similar activity profiles for the next generation antimalarials. The following review summarizes the discovery of the spiroindolones and imidazolopiperazine classes of antimalarials developed by the NGBS consortium (Novartis Institute for Tropical Diseases, Genomic Institute of the Novartis Research Foundation, Biomedical Primate Research Center, and the Swiss Tropical and Public Health Institute) currently in clinical trials.

Na(+) regulation in the malaria parasite Plasmodium falciparum involves the cation ATPase PfATP4 and is a target of the spiroindolone antimalarials.

The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na(+) influx, causing [Na(+)] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na(+)] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na(+) against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na(+)-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na(+) homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na(+) regulation. Taken together, our results are consistent with PfATP4 being a Na(+) efflux ATPase and a target of the spiroindolones.

Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design.

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein's ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.

Mechanistic study of the spiroindolones: a new class of antimalarials.

During the synthesis of the new antimalarial drug candidate NITD609, a high degree of diastereoselectivity was observed in the Pictet-Spengler reaction. By isolating both the 4E and 4Z imine intermediates, a systematic mechanistic study of the reaction under both kinetic and thermodynamic conditions was conducted. This study provides insight into the source of the diastereoselectivity for this important class of compounds.

Back to the future: lessons learned in modern target-based and whole-cell lead optimization of antimalarials.

Antimalarial drug discovery has historically benefited from the whole-cell (phenotypic) screening approach to identify lead molecules in the search for new drugs. However over the past two decades there has been a shift in the pharmaceutical industry to move away from whole-cell screening to target-based approaches. As part of a Wellcome Trust and Medicines for Malaria Venture (MMV) funded consortium to discover new blood-stage antimalarials, we used both approaches to identify new antimalarial chemotypes, two of which have progressed beyond the lead optimization phase and display excellent in vivo efficacy in mice. These two advanced series were identified through a cell-based optimization devoid of target information and in this review we summarize the advantages of this approach versus a target-based optimization. Although the each lead optimization required slightly different medicinal chemistry strategies, we observed some common issues across the different the scaffolds which could be applied to other cell based lead optimization programs.

Natural product drug discovery: the successful optimization of ISP-1 and halichondrin B.

The concept that natural products provide excellent leads for drug discovery, ultimately producing viable drugs, is a widely accepted view. Natural products embody inherent structural complexity and biological activity which often leads to new targets, pathways, or modes of action. The challenge lies in identifying quality natural product scaffolds that can ultimately result in a drug. Two recently approved drugs originating from unlikely natural product leads, ISP-1 and halichondrin B, were examples of such high quality scaffolds. In initial testing, both compounds displayed excellent in vitro potency, but more importantly were amenable to chemical optimization. This combination of unique biological activity plus the generation of structural activity relationships (SAR) may be early indicators of a high quality natural product scaffold worthy of additional studies.

Spiroindolones, a potent compound class for the treatment of malaria.

Recent reports of increased tolerance to artemisinin derivatives--the most recently adopted class of antimalarials--have prompted a need for new treatments. The spirotetrahydro-beta-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration. Spiroindolones rapidly inhibit protein synthesis in P. falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows pharmacokinetic properties compatible with once-daily oral dosing and has single-dose efficacy in a rodent malaria model.

Spirotetrahydro beta-carbolines (spiroindolones): a new class of potent and orally efficacious compounds for the treatment of malaria.

The antiplasmodial activity of a series of spirotetrahydro beta-carbolines is described. Racemic spiroazepineindole (1) was identified from a phenotypic screen on wild type Plasmodium falciparum with an in vitro IC(50) of 90 nM. Structure-activity relationships for the optimization of 1 to compound 20a (IC(50) = 0.2 nM) including the identification of the active 1R,3S enantiomer and elimination of metabolic liabilities is presented. Improvement of the pharmacokinetic profile of the series translated to exceptional oral efficacy in the P. berghei infected malaria mouse model where full cure was achieved in four of five mice with three daily doses of 30 mg/kg.

Preclinical evaluation of the antifolate QN254, 5-chloro- N'6'-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine, as an antimalarial drug candidate.

Drug resistance against dihydrofolate reductase (DHFR) inhibitors-such as pyrimethamine (PM)-has now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N'6'-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparum V1S strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparum clinical isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S) forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The drug was remarkably efficacious against Plasmodium berghei and could fully cure infected mice with three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative in vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent.