Developing kinase inhibitors for malaria: an opportunity or liability?

Highly druggable and essential to almost all aspects of cellular life, the protein and phosphoinositide kinase gene families offer a wealth of potential targets for pharmacological modulation for both noncommunicable and infectious diseases. Despite the success of kinase inhibitors in oncology and other disease indications, targeting kinases comes with significant challenges. Key hurdles for kinase drug discovery include selectivity and acquired resistance. The phosphatidylinositol 4-kinase beta inhibitor MMV390048 showed good efficacy in Phase 2a clinical trials, demonstrating the potential of kinase inhibitors for malaria treatment. Here we argue that the potential benefits of Plasmodium kinase inhibitors outweigh the risks, and we highlight the opportunity for designed polypharmacology to reduce the risk of resistance.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

The diaryl-imidazopyridazine anti-plasmodial compound, MMV652103, exhibits anti-breast cancer activity.

Breast cancer is the most common malignancy in women worldwide and it remains a global health burden, in part, due to poor response and tolerance to current therapeutics. Drug repurposing, which seeks to identify new indications for existing and investigational drugs, has become an exciting strategy to address these challenges. Here we describe the anti-breast cancer activity of a diaryl-imidazopyridazine compound, MMV652103, which was previously identified for its anti-plasmodial activity. We demonstrate that MMV652103 potently inhibits the oncogenic PI4KB and PIK3C2G lipid kinases, is selectively cytotoxic to MCF7 and T47D estrogen receptor positive breast cancer cells and inhibits their ability to survive and migrate. The underlying mechanisms involved included the induction of reactive oxygen species and activation of the DNA damage and p38 MAPK stress signaling pathways. This was associated with a G1 cell cycle arrest and an increase in levels of the cyclin-dependent kinase inhibitor p21 and activation of apoptotic and autophagic cell death pathways. Lastly, MMV652103 significantly reduced the weight and metastases of MCF7 induced tumors in an in vivo chick embryo model and displayed a favorable safety profile. These findings position MMV652103 as a promising chemotherapeutic in the treatment of oestrogen receptor positive breast cancers.

Probing the Requirements for Dual Angiotensin-Converting Enzyme C-Domain Selective/Neprilysin Inhibition.

Selective inhibition of the angiotensin-converting enzyme C-domain (cACE) and neprilysin (NEP), leaving the ACE N-domain (nACE) free to degrade bradykinin and other peptides, has the potential to provide the potent antihypertensive and cardioprotective benefits observed for nonselective dual ACE/NEP inhibitors, such as omapatrilat, without the increased risk of adverse effects. We have synthesized three 1-carboxy-3-phenylpropyl dipeptide inhibitors with nanomolar potency based on the previously reported C-domain selective ACE inhibitor lisinopril-tryptophan (LisW) to probe the structural requirements for potent dual cACE/NEP inhibition. Here we report the synthesis, enzyme kinetic data, and high-resolution crystal structures of these inhibitors bound to nACE and cACE, providing valuable insight into the factors driving potency and selectivity. Overall, these results highlight the importance of the interplay between the S1' and S2' subsites for ACE domain selectivity, providing guidance for future chemistry efforts toward the development of dual cACE/NEP inhibitors.

1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis.

Phenotypic whole cell high-throughput screening of a ∼150,000 diverse set of compounds against Mycobacterium tuberculosis (Mtb) in cholesterol-containing media identified 1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit. Structure-activity relationship (SAR) studies demonstrated a clear scope to improve whole cell potency to MIC values of <0.5 µM, and a plausible pharmacophore model was developed to describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected, suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics profiles of several active compounds from the series were established leading to the identification of a compound for in vivo efficacy proof-of-concept studies.

Plasmodium Kinases as Potential Drug Targets for Malaria: Challenges and Opportunities.

Protein and phosphoinositide kinases have been successfully exploited as drug targets in various disease areas, principally in oncology. In malaria, several protein kinases are under investigation as potential drug targets, and an inhibitor of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4KIIIß) is currently in phase 2 clinical studies. In this Perspective, we review the potential of kinases as drug targets for the treatment of malaria. Kinases are known to be readily druggable, and many are essential for parasite survival. A key challenge in the design of Plasmodium kinase inhibitors is obtaining selectivity over the corresponding human orthologue(s) and other human kinases due to the highly conserved nature of the shared ATP binding site. Notwithstanding this, there are some notable differences between the Plasmodium and human kinome that may be exploitable. There is also the potential for designed polypharmacology, where several Plasmodium kinases are inhibited by the same drug. Prior to starting the drug discovery process, it is important to carefully assess potential kinase targets to ensure that the inhibition of the desired kinase will kill the parasites in the required life-cycle stages with a sufficiently fast rate of kill. Here, we highlight key target attributes and experimental approaches to consider and summarize the progress that has been made targeting Plasmodium PI4KIIIß, cGMP-dependent protein kinase, and cyclin-dependent-like kinase 3.

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

New Amidated 3,6-Diphenylated Imidazopyridazines with Potent Antiplasmodium Activity Are Dual Inhibitors of Plasmodium Phosphatidylinositol-4-kinase and cGMP-Dependent Protein Kinase.

Recent studies on 3,6-diphenylated imidazopyridazines have demonstrated impressive in vitro activity and in vivo efficacy in mouse models of malaria infection. Herein, we report the synthesis and antiplasmodium evaluation of a new series of amidated analogues and demonstrate that these compounds potently inhibit Plasmodium phosphatidylinositol-4-kinase (PI4K) type IIIß while moderately inhibiting cyclic guanidine monophosphate (cGMP)-dependent protein kinase (PKG) activity in vitro. Using in silico docking, we predict key binding interactions for these analogues within the adenosine triphosphate (ATP)-binding site of PI4K and PKG, paving the way for structure-based optimization of imidazopyridazines targeting both Plasmodium PI4K and PKG. While several derivatives showed low nanomolar antiplasmodium activity (IC50 < 100 nM), some compounds, including piperazine analogue 28, resulted in strong dual PI4K and PKG inhibition. The compounds also demonstrated transmission-blocking potential, evident from their potent inhibition of early- and late-stage gametocytes. Finally, the current compounds generally showed improved aqueous solubility and reduced hERG (human ether-a-go-go-related gene) channel inhibition.

Dihydroartemisinin inhibits prostate cancer via JARID2/miR-7/miR-34a-dependent downregulation of Axl.

Axl expression is deregulated in several cancer types, predicts poor overall patient survival and is linked to resistance to drug therapy. Here, we evaluated a library of natural compounds for inhibitors of Axl and identified dihydroartemisinin, the active principle of the anti-malarial drug artemisinin, as an Axl-inhibitor in prostate cancer. Dihydroartemisinin blocks Axl expression leading to apoptosis, decrease in cell proliferation, migration, and tumor development of prostate cancer cells. Dihydroartemisinin treatment synergizes with docetaxel, a standard of care in metastatic prostate cancer increasing overall survival of mice with human xenografts. Dihydroartemisinin control of miR-34a and miR-7 expression leads to inhibition of Axl expression in a process at least partially dependent on regulation of chromatin via methylation of histone H3 lysine 27 residues by Jumonji, AT-rich interaction domain containing 2 (JARID2), and the enhancer of zeste homolog 2. Our discovery of a previously unidentified miR-34a/miR-7/JARID2 pathway controlling dihydroartemisinin effects on Axl expression and inhibition of cancer cell proliferation, migration, invasion, and tumor formation provides new molecular mechanistic insights into dihydroartemisinin anticancer effect on prostate cancer with potential therapeutic implications.

Insights into Integrated Lead Generation and Target Identification in Malaria and Tuberculosis Drug Discovery.

New, safe and effective drugs are urgently needed to treat and control malaria and tuberculosis, which affect millions of people annually. However, financial return on investment in the poor settings where these diseases are mostly prevalent is very minimal to support market-driven drug discovery and development. Moreover, the imminent loss of therapeutic lifespan of existing therapies due to evolution and spread of drug resistance further compounds the urgency to identify novel effective drugs. However, the advent of new public-private partnerships focused on tropical diseases and the recent release of large data sets by pharmaceutical companies on antimalarial and antituberculosis compounds derived from phenotypic whole cell high throughput screening have spurred renewed interest and opened new frontiers in malaria and tuberculosis drug discovery. This Account recaps the existing challenges facing antimalarial and antituberculosis drug discovery, including limitations associated with experimental animal models as well as biological complexities intrinsic to the causative pathogens. We enlist various highlights from a body of work within our research group aimed at identifying and characterizing new chemical leads, and navigating these challenges to contribute toward the global drug discovery and development pipeline in malaria and tuberculosis. We describe a catalogue of in-house efforts toward deriving safe and efficacious preclinical drug development candidates via cell-based medicinal chemistry optimization of phenotypic whole-cell medium and high throughput screening hits sourced from various small molecule chemical libraries. We also provide an appraisal of target-based screening, as invoked in our laboratory for mechanistic evaluation of the hits generated, with particular focus on the enzymes within the de novo pyrimidine biosynthetic and hemoglobin degradation pathways, the latter constituting a heme detoxification process and an associated cysteine protease-mediated hydrolysis of hemoglobin. We further expound on the recombinant enzyme assays, heme fractionation experiments, and genomic and chemoproteomic methods that we employed to identify Plasmodium falciparum falcipain 2 (PfFP2), hemozoin formation, phosphatidylinositol 4-kinase (PfPI4K) and Mycobacterium tuberculosis cytochrome bc1 complex as the targets of the antimalarial chalcones, pyrido[1,2-a]benzimidazoles, aminopyridines, and antimycobacterial pyrrolo[3,4-c]pyridine-1,3(2H)-diones, respectively. In conclusion, we argue for the expansion of chemical space through exploitation of privileged natural product scaffolds and diversity-oriented synthesis, as well as the broadening of druggable spaces by exploiting available protein crystal structures, -omics data, and bioinformatics infrastructure to explore hitherto untargeted spaces like lipid metabolism and protein kinases in P. falciparum. Finally, we audit the merits of both target-based and whole-cell phenotypic screening in steering antimalarial and antituberculosis chemical matter toward populating drug discovery pipelines with new lead molecules.

Antischistosomal Activity of Pyrido[1,2-a]benzimidazole Derivatives and Correlation with Inhibition of ß-Hematin Formation.

The extensive use of praziquantel against schistosomiasis raises concerns about drug resistance. New therapeutic alternatives targeting critical pathways within the parasite are therefore urgently needed. Hemozoin formation in Schistosoma presents one such target. We assessed the in vitro antischistosomal activity of pyrido[1,2-a]benzimidazoles (PBIs) and investigated correlations with their ability to inhibit ß-hematin formation. We further evaluated the in vivo efficacy of representative compounds in experimental mice and conducted pharmacokinetic analysis on the most potent. At 10 µM, 48/57 compounds resulted in >70% mortality of newly transformed schistosomula, whereas 37 of these maintained >60% mortality of adult S. mansoni. No correlations were observed between ß-hematin inhibitory and antischistosomal activities against both larval and adult parasites, suggesting possible presence of other target(s) or a mode of inhibition of crystal formation that is not adequately modeled by the assay. The most active compound in vivo showed 58.7 and 61.3% total and female worm burden reduction, respectively. Pharmacokinetic analysis suggested solubility-limited absorption and high hepatic clearance as possible contributors to the modest efficacy despite good in vitro activity. The PBIs evaluated in this report thus merit further optimization to improve their efficacy and to elucidate their possible mode of action.

Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulin-targeting antitumor agents.

The synthesis of twenty-six 4-arylcoumarin analogues of combretastatin A-4 (CA-4) led to the identification of two new compounds (25 and 26) with strong cytotoxic activity. Both compounds had a high cytotoxic effect on a CA-4-resistant colon adenocarcinoma cell line (HT29D4). The compounds affected cell cycle progression characterized by a mitotic block. The activity of these compounds against microtubules both in vitro and in cells was examined and both compounds were found to potently inhibit in vitro microtubule formation via a sub-stoichiometric mode like CA-4. By immunofluorescence, it was observed that both compounds induced strong microtubule network disruption. Our results provide a strong experimental basis to develop new potent anti-tubulin molecules targeting CA-4-resistant cancer cells.

Identification of a Potential Antimalarial Drug Candidate from a Series of 2-Aminopyrazines by Optimization of Aqueous Solubility and Potency across the Parasite Life Cycle.

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.

Identification of New Human Malaria Parasite Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors by Pharmacophore and Structure-Based Virtual Screening.

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, which the Plasmodium falciparum relies on exclusively for survival, has emerged as a promising target for antimalarial drugs. In an effort to discover new and potent PfDHODH inhibitors, 3D-QSAR pharmacophore models were developed based on the structures of known PfDHODH inhibitors and the validated Hypo1 model was used as a 3D search query for virtual screening of the National Cancer Institute database. The virtual hit compounds were further filtered based on molecular docking and Molecular Mechanics/Generalized Born Surface Area binding energy calculations. The combination of the pharmacophore and structure-based virtual screening resulted in the identification of nine new compounds that showed >25% inhibition of PfDHODH at a concentration of 10 µM, three of which exhibited IC50 values in the range of 0.38-20 µM. The most active compound, NSC336047, displayed species-selectivity for PfDHODH over human DHODH and inhibited parasite growth with an IC50 of 26 µM. In addition to this, 13 compounds inhibited parasite growth with IC50 values of ≤ 50 µM, 4 of which showed IC50 values in the range of 5-12 µM. These compounds could be further explored in the identification and development of more potent PfDHODH and parasite growth inhibitors.

New Verapamil Analogs Inhibit Intracellular Mycobacteria without Affecting the Functions of Mycobacterium-Specific T Cells.

There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5(flox/flox) (control) and ATG5(flox/flox) Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular M. tuberculosis does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on M. tuberculosis-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with M. tuberculosis whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of M. tuberculosis-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular Mycobacterium bovis BCG, and one of the three analogs (KSV21) inhibited intracellular M. tuberculosis replication at concentrations that did not inhibit M. tuberculosis-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular M. tuberculosis. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs.

A Novel Pyrazolopyridine with in Vivo Activity in Plasmodium berghei- and Plasmodium falciparum-Infected Mouse Models from Structure-Activity Relationship Studies around the Core of Recently Identified Antimalarial Imidazopyridazines.

Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure-activity relationship studies around the central core of antimalarial imidazopyridazines 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.

Structure-Activity Relationship Studies of Orally Active Antimalarial 2,4-Diamino-thienopyrimidines.

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.

Chloroquine-containing compounds: a patent review (2010 - 2014).

INTRODUCTION: Chloroquine (CQ) has been well known for its antimalarial effects since World War II. However, it is gradually being phased out from clinical use against malaria due to emergence of CQ-resistant Plasmodium falciparum strains. Besides low cost and tolerability, ongoing research has revealed interesting biochemical properties of CQ that have inspired its repurposing/repositioning in the management of various infectious/noninfectious diseases. Consequently, several novel compounds and compositions based on its scaffold have been studied and patented. AREAS COVERED: In this review, patents describing CQ and its derivatives/compositions over the last 5 years are analyzed. The review highlights the rationale, chemical structures, biological evaluation and potential therapeutic application of CQ, its derivatives and compositions. EXPERT OPINION: Repurposing efforts have dominantly focused on racemic CQ with no studies exploring the effect of the (R) and (S) enantiomers, which might potentially have additional benefits in other diseases. Additionally, evaluating other similarly acting antimalarials in clinical use and structural analogs could help maximize the intrinsic value of the 4-aminoquinolines. With regard to cancer therapy, successful repurposing of CQ-containing compounds will require linking the mode of action of these antimalarials with the signaling pathways that drive cancer cell proliferation to facilitate the development of a 4-amino-7-chloroquinoline that can be used as a synergistic partner in anticancer combination chemotherapy.

Improved antiparasitic activity by incorporation of organosilane entities into half-sandwich ruthenium(II) and rhodium(III) thiosemicarbazone complexes.

A series of ferrocenyl- and aryl-functionalised organosilane thiosemicarbazone compounds was obtained via a nucleophilic substitution reaction with an amine-terminated organosilane. The thiosemicarbazone (TSC) ligands were further reacted with either a ruthenium dimer [(η(6-i)PrC6H4Me)Ru(µ-Cl)Cl]2 or a rhodium dimer [(Cp*)Rh(µ-Cl)Cl]2 to yield a series of cationic mono- and binuclear complexes. The thiosemicarbazone ligands, as well as their metal complexes, were characterised using NMR and IR spectroscopy, and mass spectrometry. The molecular structure of the binuclear ruthenium(ii) complex was determined by single-crystal X-ray diffraction analysis. The thiosemicarbazones and their complexes were evaluated for their in vitro antiplasmodial activities against the chloroquine-sensitive (NF54) and chloroquine-resistant (Dd2) Plasmodium falciparum strains, displaying activities in the low micromolar range. Selected compounds were screened for potential ß-haematin inhibition activity, and it was found that two Rh(iii) complexes exhibited moderate to good inhibition. Furthermore, the compounds were screened for their antitrichomonal activities against the G3 Trichomonas vaginalis strain, revealing a higher percentage of growth inhibition for the ruthenium and rhodium complexes over their corresponding ligand.