Progressing the global antimalarial portfolio: Finding drugs that 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 P. 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

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 in the Pictet-Spengler reaction was observed. By isolating both of 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

Ten years of drug discovery for the developing world at the Novartis Institute for Tropical Diseases

In 2013, the Novartis Institute for Tropical Diseases (NITD) celebrated ten years of drug discovery research targeting neglected infectious diseases. Established as a public-private partnership between Novartis and the Singapore Economic Development Board, NITD was founded with the expectation that applying drug discovery expertise and cutting-edge technologies could confront the challenges of producing medicines for the developing world. In the past decade, NITD has identified two new malaria drugs now in early clinical trials, several promising leads for tuberculosis, and tool compounds for dengue fever. Key to these successes has been furthering the understanding of disease biology, applying advanced drug discovery technologies, and partnering with academic experts throughout the world

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

Abstract A new compound with potential radical cure antimalarial activity was identified using a low throughput assay with in vitro cultured Plasmodium cynomolgi hypnozoite-forms. In this assay primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exooerythrocytic development is monitored in the presence of compounds. After 6 days of culturing, the liver stage cultures are fixed and stained with anti-Hsp70 antibodies and the relative proportion of small (hypnozoite) and large (schizont) forms relative to the untreated control are counted. 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 tenfold dilutions (0.1, 1 and 10 µM final concentrations). A novel compound, designated KAI407 showed a profile similar to primaquine, efficiently killing both in vitro cultured developing liver stages and hypnozoite-forms (IC50 for small stages: were 0.69 µM (KAI407) and 0.84 µM (PQ); for developing liver stages: 0.64 µM (KAI407) and 0.37 µM (PQ)). When given as causal prophylaxis, a single oral dose of 100 mg/kg prevented blood stage parasitemia in mice. From these results we conclude that KAI407 may represent a new compound class for radical cure of P. vivax malaria

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, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells. Herein, we exploit this fact to show that the malaria parasite Plasmodium falciparum is highly sensitized to the inhibition of components of this protein quality control system. Using a variety of approaches, including activity-based protein profiling, mammalian cell- and yeast-based assays, and resistance selection, we show small molecule inhibitors of a protease component of malarial ERAD, PfSPP, simultaneously disrupt the protein’s ability to facilitate degradation of unstable proteins and inhibit its proteolytic function, resulting in lethality for the parasite. Collectively, these data validate ER quality control as a novel vulnerability for the parasite and specifically, confirm the suitability of PfSPP as an antimalarial target

Pharmacokinetics-pharmacodynamics analysis of spiroindolone analogs and KAE609 in a murine malaria model

Limited information is available on pharmacokinetic and pharmacodynamic (PK-PD) parameters driving the efficacy of anti-malarial drugs. Our objective was to determine dose response relationship to a panel of related spiroindolone analogs and identify the PK-PD index that best correlate with the efficacy of KAE609, a selected class representative. Dose-response efficacy studies were conducted in the P. berghei murine malaria model and the relationship between dose and efficacy was examined. All spiroindolone analogs studied displayed maximum parasitemia reduction with ED90 values ranging between 6 and 38 mg/kg. Most of the analogs provided a maximum survival of 14 to 15 days for the highest dose administered, with complete cure (i.e. >30 days survival) achieved only with KAE609. Further, dose fractionation studies were conducted for KAE609 and the relationship between PK-PD indices and efficacy (i.e. parasitemia reduction) was examined. PK-PD indices were calculated using as threshold (TRE) the in vitro potency against P. berghei (2*IC99). The percentage of the time in which KAE609 plasma concentration remained above 2*IC99 for 48 hrs (%T>TRE) and the AUC0-48/TRE correlated well with parasite reduction (R2=0.96 and 0.95, respectively) followed by the Cmax/TRE (R2=0.88). We cannot clearly differentiate between concentration and time-dependent killing for KAE609 based on this experiment. Furthermore, the present results suggest that for KAE609 a dosing regimen covering T>TRE of 100%, AUC/TRE of 600 and Cmax/TRE of 35 for an observation period of 48h is likely to result in >99% parasitemia reduction in the mouse

PI4K is a prophylactic, but not radical curative target in Plasmodium vivax-type malaria parasites

Two Plasmodium PI4 kinase inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical cure agents of P. 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 KDU691 and LMV599 compounds were fully protective when administered prophylactically and the more potent compound LMV599 achieved protection as a single oral 25 mg/kg dose. 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 were 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 compounds activity on established hypnozoites and predict radical cure in vivo

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 < 0.1 μM. 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

Targeting Plasmodium phosphatidylinositol-4 kinase for the treatment, prevention and elimination of malaria

Achieving the goal of malaria elimination is vitally dependent on identifying validated drug targets that are active across all stages of the Plasmodium lifecycle. Here, we identify phosphatidylinositol-4 kinase (PfPI4K) as the target of the imidazopyrazines, a novel antimalarial compound class that potently inhibits the intracellular development of multiple Plasmodium species at each stage of infection of the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in several rodent malaria models. These compounds are also active against blood-stage field isolates of the major human malaria pathogens, P. falciparum and P. vivax, and inhibit liver stage hypnozoites in the human and simian parasite P. cynomolgi. Evolved resistance, full genome-scanning and genome editing experiments in intra-erythrocytic stages as well as biochemical data, show that imidazopyrazines exert their potent antimalarial activity through interaction with the ATP-binding pocket of the lipid kinase. Inhibition of PfPI4K, alters the intracellular distribution of phosphatidylinositol-4 phosphate, the PI4K product, and interferes with cytokinesis via a Rab11A-dependent pathway. Collectively, our data define PfPI4K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify antimalarial drugs with an ideal pharmacological profile for the prevention, treatment and elimination of malaria

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis

The apicomplexan parasite Cryptosporidium is a major cause of infectious diarrhea in children and there are no consistently effective therapies to treat this disease. The search for cryptosporidiosis therapeutics has been hindered by the dearth of models amenable to modern drug discovery approaches. We established a cryptosporidiosis drug discovery screening process built on scalable phenotypic assays and a novel mouse model. We identified pyrazolopyridines as selective ATP-competitive inhibitors of the Cryptosporidium lipid kinase PI(4)K. The pyrazolopyridine KDU731 inhibits Cryptosporidium growth in multiple in vitro assays. In addition, oral treatment with KDU731 results in potent reduction in oocyst shedding in Cryptosporidium infected immunocompromised mice as well as rapid resolution of diarrheal symptoms in the neonatal calf cryptosporidiosis model. Collectively, our results chemically validate the Cryptosporidium lipid kinase PI(4)K as a drug target for the treatment of cryptosporidiosis and support the progression of the drug candidate KDU731 to further preclinical characterization