Plasmodium parasites, the causative agents of malaria, have developed resistance to most of our current antimalarial therapies, including artemisinin combination therapies which are widely described ...as our last line of defense. Antimalarial agents with a novel mode of action are urgently required. Two Plasmodium falciparum aminopeptidases, PfA-M1 and PfA-M17, play crucial roles in the erythrocytic stage of infection and have been validated as potential antimalarial targets. Using compound-bound crystal structures of both enzymes, we have used a structure-guided approach to develop a novel series of inhibitors capable of potent inhibition of both PfA-M1 and PfA-M17 activity and parasite growth in culture. Herein we describe the design, synthesis, and evaluation of a series of hydroxamic acid-based inhibitors and demonstrate the compounds to be exciting new leads for the development of novel antimalarial therapeutics.
The Plasmodium falciparum PfA-M1 and PfA-M17 metalloaminopeptidases are validated drug targets for the discovery of antimalarial agents. In order to identify dual inhibitors of both proteins, we ...developed a hierarchical virtual screening approach, followed by in vitro evaluation of the highest scoring hits. Starting from the ZINC database of purchasable compounds, sequential 3D-pharmacophore and molecular docking steps were applied to filter the virtual 'hits'. At the end of virtual screening, 12 compounds were chosen and tested against the in vitro aminopeptidase activity of both PfA-M1 and PfA-M17. Two molecules showed significant inhibitory activity (low micromolar/nanomolar range) against both proteins. Finally, the crystal structure of the most potent compound in complex with both PfA-M1 and PfA-M17 was solved, revealing the binding mode and validating our computational approach.
Malaria is a parasitic disease that remains a global health burden. The ability of the parasite to rapidly develop resistance to therapeutics drives an urgent need for the delivery of new drugs. The ...Medicines for Malaria Venture have compounds known for their antimalarial activity, but not necessarily the molecular targets. In this study, we assess the ability of the "MMV 400" compounds to inhibit the activity of three metalloaminopeptidases from Plasmodium falciparum, PfA-M1, PfA-M17 and PfM18 AAP. We have developed a multiplex assay system to allow rapid primary screening of compounds against all three metalloaminopeptidases, followed by detailed analysis of promising compounds. Our results show that there were no PfM18AAP inhibitors, whereas two moderate inhibitors of the neutral aminopeptidases PfA-M1 and PfA-M17 were identified. Further investigation through structure-activity relationship studies and molecular docking suggest that these compounds are competitive inhibitors with novel binding mechanisms, acting through either non-classical zinc coordination or independently of zinc binding altogether. Although it is unlikely that inhibition of PfA-M1 and/or PfA-M17 is the primary mechanism responsible for the antiplasmodial activity reported for these compounds, their detailed characterization, as presented in this work, pave the way for their further optimization as a novel class of dual PfA-M1/PfA-M17 inhibitors utilising non-classical zinc binding groups.
To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound
is a selective, nanomolar inhibitor ...of both
and
aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites.
can kill sexual-stage
, is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials.
-resistant
exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm
as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as
, and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to
appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial.
Despite a century of control and eradication campaigns, malaria remains one of the world's most devastating diseases. Our once-powerful therapeutic weapons are losing the war against the Plasmodium ...parasite, whose ability to rapidly develop and spread drug resistance hamper past and present malaria-control efforts. Finding new and effective treatments for malaria is now a top global health priority, fuelling an increase in funding and promoting open-source collaborations between researchers and pharmaceutical consortia around the world. The result of this is rapid advances in drug discovery approaches and technologies, with three major methods for antimalarial drug development emerging: (i) chemistry-based, (ii) target-based, and (iii) cell-based. Common to all three of these approaches is the unique ability of structural biology to inform and accelerate drug development. Where possible, SBDD (structure-based drug discovery) is a foundation for antimalarial drug development programmes, and has been invaluable to the development of a number of current pre-clinical and clinical candidates. However, as we expand our understanding of the malarial life cycle and mechanisms of resistance development, SBDD as a field must continue to evolve in order to develop compounds that adhere to the ideal characteristics for novel antimalarial therapeutics and to avoid high attrition rates pre- and post-clinic. In the present review, we aim to examine the contribution that SBDD has made to current antimalarial drug development efforts, covering hit discovery to lead optimization and prevention of parasite resistance. Finally, the potential for structural biology, particularly high-throughput structural genomics programmes, to identify future targets for drug discovery are discussed.
The family of M17 aminopeptidases (alias ‘leucine aminopeptidases’, M17-LAPs) utilize a highly conserved hexameric structure and a binuclear metal center to selectively remove N-terminal amino acids ...from short peptides. However, M17-LAPs are responsible for a wide variety of functions that are seemingly unrelated to proteolysis. Herein, we aimed to investigate the myriad of functions attributed to M17. Further, we attempted to differentiate between the different molecular mechanisms that allow the conserved hexameric structure of an M17-LAP to mediate such diverse functions. We have provided an overview of research that identifies precise physiological roles of M17-LAPs, and the distinct mechanisms by which the enzymes moderate those roles. The review shows that the conserved hexameric structure of the M17-LAPs has an extraordinary capability to moderate different molecular mechanisms. We have broadly categorized these mechanisms as ‘aminopeptidase-based’, which include the characteristic proteolysis reactions, and ‘association-driven’, which involves moderation of the molecule's macromolecular assembly and higher order complexation events. The different molecular mechanisms are capable of eliciting very different cellular outcomes, and must be regarded as distinct when the physiological roles of this large and important family are considered.
•M17 aminopeptidases possess a highly conserved structure and active site.•They are multifunctional, capable of performing diverse functions far beyond peptide hydrolysis.•M17 aminopeptidases from plants possess chaperone activity, while in bacteria they can bind DNA.•Here we review mechanisms that underpin the multifunctional nature of the M17 aminopeptidases.
M1 aminopeptidase enzymes are a diverse family of metalloenzymes characterized by conserved structure and reaction specificity. Excluding viruses, M1 aminopeptidases are distributed throughout all ...phyla, and have been implicated in a wide range of functions including cell maintenance, growth and development, and defense. The structure and catalytic mechanism of M1 aminopeptidases are well understood, and make them ideal candidates for the design of small‐molecule inhibitors. As a result, many research groups have assessed their utility as therapeutic targets for both infectious and chronic diseases of humans, and many inhibitors with a range of target specificities and potential therapeutic applications have been developed. Herein, we have aimed to address these studies, to determine whether the family of M1 aminopeptidases does in fact present a universal target for the treatment of a diverse range of human diseases. Our analysis indicates that early validation of M1 aminopeptidases as therapeutic targets is often overlooked, which prevents the enzymes from being confirmed as drug targets. This validation cannot be neglected, and needs to include a thorough characterization of enzymes’ specific roles within complex physiological pathways. Furthermore, any chemical probes used in target validation must be carefully designed to ensure that specificity over the closely related enzymes has been achieved. While many drug discovery programs that target M1 aminopeptidases remain in their infancy, certain inhibitors have shown promise for the treatment of a range of conditions including malaria, hypertension, and cancer.
The large family of therapeutically interesting M1 aminopeptidase enzymes has enormous potential to provide important drug targets. However, conservation of sequence, structure, and substrate specificity results in substantial challenges, and necessitates the need for careful target validation. Some studies have achieved this, and successfully validated M1 aminopeptidases as targets for the treatment of malaria, hypertension, and cancer.
Aminopeptidase N (APN/CD13) is a zinc‐dependent ubiquitous transmembrane ectoenzyme that is widely present in different types of cells. APN is one of the most extensively studied ...metalloaminopeptidases as an anti‐cancer target due to its significant role in the regulation of metastasis and angiogenesis. Previously, we identified a potent and selective APN inhibitor, N‐(2‐(Hydroxyamino)‐2‐oxo‐1‐(3′,4′,5′‐trifluoro‐1,1′‐biphenyl‐4‐yl)ethyl)‐4‐(methylsulfonamido)benzamide (3). Herein, we report the further modifications performed to explore SAR around the S1 subsite of APN and to improve the physicochemical properties. A series of hydroxamic acid analogues were synthesised, and the pharmacological activities were evaluated in vitro. N‐(1‐(3′‐Fluoro‐1,1′‐biphenyl‐4‐yl)‐2‐(hydroxyamino)‐2‐oxoethyl)‐4‐(methylsulfonamido)benzamide (6 f) was found to display an extremely potent inhibitory activity in the sub‐nanomolar range.
Targeting metastasis and angiogenesis: Hydroxamic acid bearing small molecules were synthesised as potent inhibitors of aminopeptidase N (APN) for the treatment of cancer. The 3,4,5‐trifluorophenyl group of the lead compound 3 was replaced with various substituents to improve both potency and solubility. The SAR results indicated 3‐fluorophenyl analogue 6 f to be the most potent APN inhibitor that showed inhibitory activity in the sub‐nanomolar range.
M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and ...Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel antimalarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryoelectron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer ↔ dimer ↔ tetramer ↔ hexamer, which becomes directional toward the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.
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