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Fast-acting small molecules targeting malarial aspartyl proteases, plasmepsins, inhibit malaria infection at multiple life stages Snigdha Singh, Vinoth Rajendran, Jiang He, Amit Kumar Singh, Angela O Achieng, Vandana Kumari, Akansha Pant, Armiyaw S Nasamu, Mansi Pandit, Jyoti Singh, Afshana Quadiri, Nikesh Gupta, Poonam Singh, Prahlad C Ghosh, Brajendra Kumar Singh, Latha Narayanan, Prakasha Kempaiah, Ramesh Chandra, Ben M Dunn, Kailash C Pandey, Daniel E Goldberg, Agam P Singh, and Brijesh Rathi ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00197 • Publication Date (Web): 17 Dec 2018 Downloaded from http://pubs.acs.org on December 17, 2018
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Fast-acting small molecules targeting malarial aspartyl proteases, plasmepsins, inhibit malaria infection at multiple life stages Snigdha Singh1,2,±, Vinoth Rajendran3,±, Jiang He4,±, Amit K Singh2, Angela O. Achieng5, Vandana Kumari6, Akansha Pant6, Armiyaw S. Nasamu7, Mansi Pandit8, Jyoti Singh9, Afshana Quadiri9, Nikesh Gupta10, Poonam11, Prahlad C Ghosh3, Brajendra K Singh2, Latha Narayanan8, Prakasha Kempaiah5,12, Ramesh Chandra2, Ben M Dunn13, Kailash C Pandey6,14, Daniel E Goldberg7, Agam P Singh9,* and Brijesh Rathi1,15,* 1Laboratory
for Translational Chemistry and Drug Discovery, Department of Chemistry, Hansraj College University Enclave, University of Delhi, Delhi-110007 India 2Department of Chemistry, University of Delhi, Delhi-110007 India 3Department of Biochemistry, University of Delhi South Campus, New Delhi-110021 India 4Institute for Medical Engineering and Science, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 5Center
for Global Health, Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, United States of America 6National Institute of Malaria Research, Host-Parasite Interaction Biology Group, Lab. No. 219, Sector-8 Dwarka, New Delhi 110077, India 7Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA 8Bioinformatics
Infrastructure Facility, Sri Venkateswara College, University of Delhi South Campus, New Delhi 110 021, India 9Infectious Diseases Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India 10Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi-110067, India 11Department of Chemistry, Miranda House, University of Delhi North Campus, Delhi110007, India 12Department of Medicine, Loyola University Stritch School of Medicine, 2160 South 1st Avenue, Chicago, IL 60153, USA 13Department of Biochemistry & Molecular Biology, University of Florida College of Medicine, P.O. Box 100245, Gainesville, FL, United States of America 14Department
of Biochemistry, National Institute for Research in Environmental Health, ICMR, Bhopal, India 15Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 Unites States of America ±These
authors contributed equally
Corresponding Author Brijesh Rathi:
[email protected] Agam P Singh:
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The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of P. falciparum and P. vivax. New, effective and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we
synthesized
a
set
of
novel
hydroxyethylamines and investigated their activities in vitro and in vivo. All the compounds tested had an inhibitory effect on the blood stage of P. falciparum at sub-micromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant P. berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stage (III-V) of P. falciparum in culture and liver stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.
Key words: Multi-stage malaria inhibitor, Plasmepsins, Phthalimide, Hydroxyethylamine, Marked-killing inhibitor.
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Malaria, caused by the infection of the protozoan parasite Plasmodium, is a devastating disease that affects millions of lives each year1. The malaria infection cycle involves two hosts: the human and Anopheles mosquito. During a blood meal, the infected female mosquito inoculates sporozoites under the skin of the human host, which then travel to the liver, infect the hepatocytes, and begin the asymptomatic development into schizonts before getting released as merosomes containing thousands of merozoites to initiate the blood stage infection2. As the mosquito takes the blood meal from an infected human host, the gametocytes are ingested and the sexual stage of development occurs in the mosquito gut, ultimately forming oocysts that further develop into sporozoites2. The complex life cycle of malaria offers several opportunities for intervention. Efforts to eradicate malaria include insecticide-treated bed nets, insecticide sprays, and artemisinin-based combinational treatments (ACT)3-5, non-artemisinin-based partner drugs6, which have considerably reduced malaria incidences and mortality. However, due to the increasing emergence of resistance to the frontline therapies such as artemisinin7-10, the prevalence of Plasmodium infection persists, with continued ACT failure in endemic areas such as Thailand-Cambodia border11,12. Furthermore, clinically used non-artemisinin drugs have shown decreased effectiveness to P. falciparum with rapid development of resistance13-15. Finally, the only licensed RTS,S vaccine Mosquirix has limited protection rate, with efficacy of about 30%, highlighting the need to develop better antimalarials16. Most existing antimalarial drugs target narrow range of the parasite’s life processes, mainly the asexual blood stage that underlies the disease’s clinical symptoms17. With drug-resistant Plasmodium strains evolving18, 19, novel therapeutics against different molecular targets in the blood stage parasites are urgently needed. Inhibitors which can simultaneously target both the blood and liver stage of infection, offer alternative opportunities to reduce the proliferation and transmission burden of the parasite in the human host19-23. Specifically, as liver stage is an obligatory developmental step that precedes the symptomatic blood stage, and one during which the parasite number increases exponentially, it represents an attractive bottleneck step vulnerable for intervention for different malaria strains. There have been substantial efforts to develop antimalarials to inhibit the liver stage infection of P. vivax and P. falciparum24,25. However, a limited number of drugs target the liver stage infection, and despite ongoing efforts to look for new therapeutics, few have progressed to clinical trials26, 27. The paucity of novel antimalarial drugs against other stages of malaria infection demands more efforts to screen for compounds that may be active against Plasmodium at single or multiple stages of
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the infection cycle28. Based on recent evidence, compounds such as atovaquone, pyronaridine, quinolones, imidazolopiperazines, pyrazines, azetidines, and xenomycins have all exhibited the capacity to inhibit several life stages of the malaria parasite18,
29-38,
prompting us to further look for chemical moieties with similar effects. Previously in our laboratory, we synthesized a set of hydroxyethylamine-phthalimide (HEAPht) analogs that showed antimalarial activity against blood-stage parasites39. Preliminary studies indicated that these compounds target plasmepsins (Plms) II and IV, two malarial aspartyl proteases found in the digestive vacuole of the parasite. Plms are critical for hemoglobin catabolism in the erythrocytic stage of the parasite and are recognized as novel potential targets for new antimalarial therapy40-45. For these HEA-Pht analogs, the combined presence of HEA and cyclic amines (piperazines and piperidines) was found to be crucial for the antimalarial activity, and the scaffolds of these compounds are prone to further chemical modifications39. Prompted by these initial characterizations, we hypothesized that further modifications could yield more potent compounds against the blood stage, and even lead to molecules inhibiting multiple life stages of the parasite, as plasmepsins are expressed across all stages of the parasite’s life cycle40-45. In the current work, we synthesized six compounds based on the core chemical scaffold of HEA-Pht analogs and evaluated their activity against multiple stages of malaria infection, including the asexual blood stage, liver stage, and gametocyte stage of the parasite life cycle. Furthermore, we characterized their interactions with plasmepsins and antimalarial profile alone or in combination therapy for malaria treatment. RESULTS AND DISCUSSION Chemical synthesis of HEA-Pht based compounds, assessment of their antimalarial activity and cytotoxicity during blood stage Previously, our group found that chemical entities comprising scaffolds viz. piperazine, HEA39 and Pht46 work as potential antimalarial agents, albeit not all exhibiting strong inhibitory effects. HEA-Pht based molecules were identified as potent inhibitors of Plm II and IV, two malarial aspartyl proteases found to be expressed across different stages of malaria infection cycle43-45. To develop more potent compounds, we synthesized C2 symmetric HEA-Pht analogs without significantly perturbing their core scaffolds. These modifications aimed to improve the compounds’ antimalarial activity profile, particularly against multiple life stages of the malaria parasite. Six new analogs, compounds (1-6), with
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or without the Pht scaffold, a molecular flexibility by adopting two different types of compounds (Fig. 1A), were synthesized (Sup Fig. S1). The methyl group on Pht was included to potentially increase the binding affinity with the aspartyl proteases as it modestly extends the Pht scaffold for stronger electrostatic interactions, while phenylalanine39 and leucine46 were selected as linkers due to their vital antimalarial role for obtaining the maximal activity and minimal cytotoxicity. To obtain a general performance profile of these newly synthesized compounds, we first sought to measure their activity against the growth of drug-susceptible P. falciparum (Pf3D7) at the blood stage in asynchronized culture, as done previously by our group39. Infected erythrocytes were treated with various concentrations of compounds (1-6) and incubated at 37 oC for 48 h, and the parasite viability was measured by a [3H] hypoxanthine incorporation assay. As shown (Fig. 1B), all six compounds displayed growth inhibition effect for Pf3D7 with IC50 values less than 6 µM. Among them, compounds 2 and 5 were most potent, with IC50 values at 1.141.10 µM and 1.310.72 µM, respectively. Based on the inhibition profile, compound 2 and 5 were selected as lead molecules in this study for further investigations. To test compound 2 and 5’s selectivity and cytotoxic effect, proliferation inhibition assays were performed against two additional strains: drug-susceptible (PfD6) and drug-resistant P. falciparum (PfDd2), and similarly, we found both compounds 2 and 5 exhibiting potent activity against PfD6 and PfDd2 (Fig. 1C and 1D). The inhibitory effect did not seem to stem from non-specific effects caused by both compounds, as we observed little cytotoxicity of compound 2 and 5 in peripheral blood mononuclear cells (PBMC) and leukemic monocytic cell lines (U937) (Fig. 1C). The 50% cytotoxic concentrations (CC50) in PBMC for compound 2 were at ~ 5µ M and 5 at ~11.1 µM, while CC50 in U937 cells were around 20 µM. We also determined the selectivity of these compounds for Plasmodium in comparison to PBMC and U937 cells. The selectivity indexes (SI) showed that both the compounds were selective against the parasite, with SI values ranging from 4 to 40. In contrast, compound 6, which had an IC50 value of 1.350.58 µM, exhibited a lower selectivity index (SI
