Current and emerging target identification methods for novel antimalarials.

New antimalarial compounds with novel mechanisms of action are urgently needed to combat the recent rise in antimalarial drug resistance. Phenotypic high-throughput screens have proven to be a successful method for identifying new compounds, however, do not provide mechanistic information about the molecular target(s) responsible for antimalarial action. Current and emerging target identification methods such as in vitro resistance generation, metabolomics screening, chemoproteomic approaches and biophysical assays measuring protein stability across the whole proteome have successfully identified novel drug targets. This review provides an overview of these techniques, comparing their strengths and weaknesses and how they can be utilised for antimalarial target identification.

1,3-Benzodioxole-Modified Noscapine Analogues: Synthesis, Antiproliferative Activity, and Tubulin-Bound Structure.

Since the revelation of noscapine's weak anti-mitotic activity, extensive research has been conducted over the past two decades, with the goal of discovering noscapine derivatives with improved potency. To date, noscapine has been explored at the 1, 7, 6', and 9'-positions, though the 1,3-benzodioxole motif in the noscapine scaffold that remains unexplored. The present investigation describes the design, synthesis and pharmacological evaluation of noscapine analogues consisting of modifications to the 1,3-benzodioxole moiety. This includes expansion of the dioxolane ring and inclusion of metabolically robust deuterium and fluorine atoms. Favourable structural modifications were subsequently incorporated into multi-functionalised noscapine derivatives that also possessed modifications previously shown to promote anti-proliferative activity in the 1-, 6'- and 9'-positions. Our research efforts afforded the deuterated noscapine derivative 14 e and the dioxino-containing analogue 20 as potent cytotoxic agents with EC50 values of 1.50 and 0.73 µM, respectively, against breast cancer (MCF-7) cells. Compound 20 also exhibited EC50 values of <2 µM against melanoma, non-small cell lung carcinoma, and cancers of the brain, kidney and breast in an NCI screen. Furthermore, compounds 14 e and 20 inhibit tubulin polymerisation and are not vulnerable to the overexpression of resistance conferring P-gp efflux pumps in drug-resistant breast cancer cells (NCIADR/RES ). We also conducted X-ray crystallography studies that yielded the high-resolution structure of 14 e bound to tubulin. Our structural analysis revealed the key interactions between this noscapinoid and tubulin and will assist with the future design of noscapine derivatives with improved properties.

Discovery and development of 2-aminobenzimidazoles as potent antimalarials.

The emergence of Plasmodium falciparum resistance to frontline antimalarials, including artemisinin combination therapies, highlights the need for new molecules that act via novel mechanisms of action. Herein, we report the design, synthesis and antimalarial activity of a series of 2-aminobenzimidazoles, featuring a phenol moiety that is crucial to the pharmacophore. Two potent molecules exhibited IC50 values against P. falciparum 3D7 strain of 42 ± 4 (3c) and 43 ± 2 nM (3g), and high potency against strains resistant to chloroquine (Dd2), artemisinin (Cam3.IIC580Y) and PfATP4 inhibitors (SJ557733), while demonstrating no cytotoxicity against human cells (HEK293, IC50 > 50 µM). The most potent molecule, possessing a 4,5-dimethyl substituted phenol (3r) displayed an IC50 value of 6.4 ± 0.5 nM against P. falciparum 3D7, representing a 12-fold increase in activity from the parent molecule. The 2-aminobenzimidazoles containing a N1-substituted phenol represent a new class of molecules that have high potency in vitro against P. falciparum malaria and low cytotoxicity. They possessed attractive pharmaceutical properties, including low molecular weight, high ligand efficiency, high solubility, synthetic tractability and low in vitro clearance in human liver microsomes.

Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound, JPC-3210, Against Plasmodium falciparum.

The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a "multi-omics" workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro ß-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of ß-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

A Novel Class of N-Sulfonyl and N-Sulfamoyl Noscapine Derivatives that Promote Mitotic Arrest in Cancer Cells.

Noscapine displays weak anticancer efficacy and numerous research efforts have attempted to generate more potent noscapine analogues. These modifications included the replacement of the N-methyl group in the 6'-position with a range of substituents, where N-ethylcarbamoyl substitution was observed to possess enhanced anticancer activity. Herein, we describe advances in this area, namely the synthesis and pharmacological evaluation of a series of N-sulfonyl and N-sulfamoyl noscapine derivatives. A number of these sulfonyl-containing noscapinoids demonstrated improved activities compared to noscapine. ((R)-5-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-((1-methyl-1H-imidazol-4-yl)sulfonyl)-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline) (14 q) displayed sub-micromolar activities of 560, 980, 271 and 443 nM against MCF-7, PANC-1, MDA-MB-435 and SK-MEL-5 cells, respectively. This antiproliferative effect was also maintained against drug-resistant NCI/AdrRES cells despite high expression of the multidrug efflux pump, P-glycoprotein.

The Development Process for Discovery and Clinical Advancement of Modern Antimalarials.

Malaria is a devastating disease caused by Plasmodium parasites, resulting in approximately 435000 deaths in 2018. The impact of malaria is compounded by the emergence of widespread resistance to current antimalarial therapies. Recently, a new strategy was initiated to screen small molecule collections against the Plasmodium parasite enabling the identification of new antimalarial chemotypes with novel modes of action. This initiative ushered in the modern era of antimalarial drug development, and as a result, numerous lead candidates are advancing toward or are currently in human clinical trials. In this Perspective, we describe the development pathway of four of the most clinically advanced modern antimalarials, KAE609, KAF156, DSM265, and MMV048. Additionally, the mechanism of action and life-cycle stage specificity of the four antimalarials is discussed in relation to aligning with global strategies to treat and eliminate malaria. This perspective serves as a guide to the expectations of modern antimalarial drug development.

Identification of the Binding Site of Apical Membrane Antigen†1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Synthesis and Pharmacological Evaluation of Noscapine-Inspired 5-Substituted Tetrahydroisoquinolines as Cytotoxic Agents.

A series of 5-substituted tetrahydroisoquinolines was synthesized via a 10-step linear synthesis to assess whether replacement of noscapine's southern isobenzofuranone with other moieties resulted in retained cytotoxic activity. One such molecule, 18g, bearing a para-methoxybenzyl functionality with N-ethylcarbamoyl substitution, produced cell-cycle arrest at the G2/M phase with an EC50 of 2.7 µM in the MCF-7 breast-cancer cell line, a 7-fold increase compared with that of noscapine (5). This molecule had similar activity (EC50 of 2.5 µM) against the resistant NCI/AdrRES cell line, demonstrating its potential to overcome or avert known resistance mechanisms, unlike current cytotoxic agents. Compound 18g was found to modify the drug-efflux activity of P-gp and, in combination studies, potentiate the antiproliferative activity of vinblastine. These results provide insights into structural modifications to noscapine that will guide future development toward more potent cytotoxic agents that are active against resistant cancer cells.

A Structure-Activity Relationship Study of Bitopic N6-Substituted Adenosine Derivatives as Biased Adenosine A1 Receptor Agonists.

The adenosine A1 receptor (A1AR) is a potential novel therapeutic target for myocardial ischemia-reperfusion injury. However, to date, clinical translation of prototypical A1AR agonists has been hindered due to dose limiting adverse effects. Recently, we demonstrated that the biased bitopic agonist 1, consisting of an adenosine pharmacophore linked to an allosteric moiety, could stimulate cardioprotective A1AR signaling in the absence of unwanted bradycardia. Therefore, this study aimed to investigate the structure-activity relationship of compound 1 biased agonism. A series of novel derivatives of 1 were synthesized and pharmacologically profiled. Modifications were made to the orthosteric adenosine pharmacophore, linker, and allosteric 2-amino-3-benzoylthiophene pharmacophore to probe the structure-activity relationships, particularly in terms of biased signaling, as well as A1AR activity and subtype selectivity. Collectively, our findings demonstrate that the allosteric moiety, particularly the 4-(trifluoromethyl)phenyl substituent of the thiophene scaffold, is important in conferring bitopic ligand bias at the A1AR.

Antimalarial drug discovery targeting apical membrane antigen 1.

Malaria continues to frustrate humanity's attempts to eradicate this deadly disease. Although gains have been made over the last 15 years, drug resistance to malaria continues to be a major concern. The lack of new antimalarials with novel mechanisms of action continues to challenge the scientific community to find innovative targets to combat this persistent disease. One such target, apical membrane antigen 1 (AMA1), is an essential protein that helps the parasite invade host erythrocytes. Recently, a number of efforts have focused on the druggability of this target, aiming to block the interactions of AMA1 that mediate invasion of host cells. This review covers recent progress in drug discovery targeting this crucial protein-protein interaction in malaria.

VCP746, a novel A1 adenosine receptor biased agonist, reduces hypertrophy in a rat neonatal cardiac myocyte model.

VCP746 is a novel A1 adenosine receptor (A1 AR) biased agonist previously shown to be cytoprotective with no effect on heart rate. The aim of this study was to investigate the potential anti-hypertrophic effect of VCP746 in neonatal rat cardiac myocytes (NCM). NCM hypertrophy was stimulated with interleukin (IL)-1ß (10 ng/mL), tumour necrosis factor (TNF)-α (10 ng/mL) or Ang II (100 nmol/L) and was assessed by (3) H-leucine incorporation assay. VCP746 significantly inhibited IL-1ß-, TNF-α- and Ang II-stimulated NCM hypertrophy as determined by (3) H-leucine incorporation. The anti-hypertrophic effect of VCP746 was also more potent than that of the prototypical A1 AR agonist, N(6) -cyclopentyladenosine (CPA). Further investigation with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay showed that neither CPA nor VCP746 had any effect on cell viability, confirming that the reduction in (3) H-leucine incorporation mediated by CPA and VCP746 was not due to a reduction in cell viability. IL-1ß, TNF-α and Ang II were also shown to increase the mRNA expression of hypertrophy biomarkers, ANP, ß-MHC and α-SKA in NCM. Treatment with VCP746 at concentrations as low as 1 nmol/L suppressed mRNA expression of ANP, ß-MHC and α-SKA stimulated by IL-1ß, TNF-α or Ang II, demonstrating the broad mechanistic basis of the potent anti-hypertrophic effect of VCP746. This study has shown that the novel A1 AR agonist, VCP746, is able to attenuate cardiac myocyte hypertrophy. As such, VCP746 is potentially useful as a pharmacological agent in attenuating cardiac remodelling, especially in the post-myocardial infarction setting, given its previously established cytoprotective properties.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

Design, Synthesis, and Biological Evaluation of Tetra-Substituted Thiophenes as Inhibitors of p38α MAPK.

p38α mitogen-activated protein kinase (MAPK) plays a role in several cellular processes and consequently has been a therapeutic target in inflammatory diseases, cancer, and cardiovascular disease. A number of known p38α MAPK inhibitors contain vicinal 4-fluorophenyl/4-pyridyl rings connected to either a 5- or 6-membered heterocycle. In this study, a small library of substituted thiophene-based compounds bearing the vicinal 4-fluorophenyl/4-pyridyl rings was designed using computational docking as a visualisation tool. Compounds were synthesised and evaluated in a fluorescence polarisation binding assay. The synthesised analogues had a higher binding affinity to the active phosphorylated form of p38α MAPK than the inactive nonphosphorylated form of the protein. 4-(2-(4-fluorophenyl)thiophen-3-yl)pyridine had a K i value of 0.6 µm to active p38α MAPK highlighting that substitution of the core ring to a thiophene retains affinity to the enzyme and can be utilised in p38α MAPK inhibitors. This compound was further elaborated using a substituted phenyl ring in order to probe the second hydrophobic pocket. Many of these analogues exhibited low micromolar affinity to active p38α MAPK. The suppression of neonatal rat fibroblast collagen synthesis was also observed suggesting that further development of these compounds may lead to potential therapeutics having cardioprotective properties.

Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

Structure and dynamics of apical membrane antigen 1 from Plasmodium falciparum FVO.

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from Plasmodium falciparum strain FVO. We compare our new structure to those of AMA1 from P. falciparum 3D7 and Plasmodium vivax. A combination of normalized B factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting P. falciparum AMA1.

Ligand-induced conformational change of Plasmodium falciparum AMA1 detected using 19F NMR.

We established an efficient means of probing ligand-induced conformational change in the malaria drug target AMA1 using 19F NMR. AMA1 was labeled with 5-fluorotryptophan (5F-Trp), and the resulting 5F-Trp resonances were assigned by mutagenesis of the native Trp residues. By introducing additional Trp residues at strategic sites within a ligand-responsive loop, we detected distinct conformational consequences when various peptide and small-molecule ligands bound AMA1. Our results demonstrate an increase in flexibility in this loop caused by the native ligand, as inferred from, but not directly observed in, crystal structures. In addition, we found evidence for long-range allosteric changes in AMA1 that are not observed crystallographically. This method will be valuable in ongoing efforts to identify and characterize therapeutically relevant inhibitors of protein-protein interactions involving AMA1 and is generalizable to the study of ligand-induced conformational change in a wide range of other drug targets.

Molecular mechanisms of bitopic ligand engagement with the M1 muscarinic acetylcholine receptor.

TBPB and 77-LH-28-1 are selective agonists of the M1 muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M1 mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.

An allosteric modulator of the adenosine A1 receptor improves cardiac function following ischaemia in murine isolated hearts.

The effect of an allosteric modulator of the adenosine A1 receptors was investigated using an ischaemia-reperfusion protocol in murine isolated hearts. Isolated hearts were perfused with Kreb-Henseleit solution gassed with carbogen gas (95% O2 and 5% CO2) in Langendorff mode and electrically paced at 480 bpm. Following 20 min equilibration and 20 min global normothermic ischaemia, the allosteric modulator VCP333 (1 µM) or the adenosine A1 receptor partial agonist VCP102 (10 µM) were infused after 5 min of reperfusion for 15 min. Upon termination of the drug treatment, reperfusion continued for a further 40 min. At the end of 60 min reperfusion, treatment with VCP333 or VCP102 improved the recovery of the left ventricular developed pressure when compared to control group responses (p < 0.05). Neither compound affected end diastolic pressure, coronary flow rates or dP/dtmax values when compared to control tissues during reperfusion (p > 0.05). The infusion of VCP102 or VCP333 during reperfusion reduced cardiac troponin I efflux to 6.7% and 25% respectively of control heart efflux (p < 0.05). This data indicates that the allosteric modulator of the adenosine A1 receptor (VCP333) has similar characteristics to the adenosine receptor partial agonist VCP102 as it improves cardiac function and reduces myocardial cell death following an ischaemic episode.

Reverse engineering of the selective agonist TBPB unveils both orthosteric and allosteric modes of action at the M1 muscarinic acetylcholine receptor.

Recent interest in the M1 muscarinic acetylcholine (ACh) receptor (mAChR) has led to the discovery of various selective agonists for the receptor. The novel selective agonist 1-(1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (TBPB) displays unprecedented functional selectivity at the M1 mAChR. This functional selectivity has been described to stem from sole interaction with an allosteric site, although the evidence for such a mechanism is equivocal. To delineate TBPB's mechanism of action, several truncated variants of TBPB were synthesized and characterized. Binding experiments with [³H]N-methylscopolamine at the M1, M2, M3, and M4 mAChRs revealed radioligand displacement in a manner consistent with a competitive binding mode at the orthosteric site by TBPB and fragment derivatives. Cell-based functional assays of fragment derivatives of TBPB identified both agonistic and antagonistic moieties, one of which, 1-(1-cyclohexylpiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (VCP794), lost agonistic selectivity for the M1 mAChR. Further interaction experiments between TBPB or its antagonist fragments with ACh also indicated a mechanism consistent with competitive binding at mAChRs. However, interaction with an allosteric site by an antagonist fragment of TBPB was demonstrated via its ability to retard radioligand dissociation. To reconcile this dual orthosteric/allosteric pharmacological behavior, we propose that TBPB is a bitopic ligand, interacting with both the orthosteric site and an allosteric site, at the M1 mAChR. This mechanism may also be the case for other selective agonists for mAChRs, and should be taken into consideration in the profiling and classification of new novel selective agonists for this receptor family.

Synthesis and pharmacological evaluation of dual acting antioxidant A(2A) adenosine receptor agonists.

A series of adenosine-5'-N-alkylcarboxamides and N(6)-(2,2-diphenylethyl)adenosine-5'-N-alkylcarboxamides bearing antioxidant moieties in the 2-position were synthesized from the versatile intermediate, O(6)-(benzotriazol-1-yl)-2-fluoro-2',3'-O-isopropylideneinosine-5'-N-alkylcarboxamide (1). These compounds were evaluated as A(2A) adenosine receptor (A(2A)R) agonists in a cAMP accumulation assay, and a number of potent and selective agonists were identified. Three of these compounds were evaluated further in an ischemic injury cell survival assay and a reactive oxygen species (ROS) production assay whereby 15b and 15c were shown to reduce ROS activity and cell death due to ischemia.