M1 Alanyl Aminopeptidase Research Articles

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Antiplasmodial Activity Evaluation of a Bestatin-Related Aminopeptidase Inhibitor, Phebestin.

The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 μM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.

Overexpression of Plasmodium falciparum M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development.

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in P. falciparum.

Biochemical and cellular characterisation of the Plasmodium falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP)

The Plasmodium falciparum M1 alanyl aminopeptidase and M17 leucyl aminopeptidase, PfM1AAP and PfM17LAP, are potential targets for novel anti-malarial drug development. Inhibitors of these aminopeptidases have been shown to kill malaria parasites in culture and reduce parasite growth in murine models. The two enzymes may function in the terminal stages of haemoglobin digestion, providing free amino acids for protein synthesis by the rapidly growing intra-erythrocytic parasites. Here we have performed a comparative cellular and biochemical characterisation of the two enzymes. Cell fractionation and immunolocalisation studies reveal that both enzymes are associated with the soluble cytosolic fraction of the parasite, with no evidence that they are present within other compartments, such as the digestive vacuole (DV). Enzyme kinetic studies show that the optimal pH of both enzymes is in the neutral range (pH 7.0–8.0), although PfM1AAP also possesses some activity (< 20%) at the lower pH range of 5.0–5.5. The data supports the proposal that PfM1AAP and PfM17LAP function in the cytoplasm of the parasite, likely in the degradation of haemoglobin-derived peptides generated in the DV and transported to the cytosol.

Steered molecular dynamics simulations reveal critical residues for (un)binding of substrates, inhibitors and a product to the malarial M1 aminopeptidase.

Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the ‘water reservoir’, (III) ligand reorientation in the ‘water reservoir’ and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site.

KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC50=18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.

Steered Molecular Dynamic Simulations Reveal Critical Residues for (Un)Binding of Substrates, Inhibitors and a Product of the Malarial PFM1AAP

Malaria is one of the most prevalent and fatal infectious diseases of the world and is compounded by wide spread drug resistance, with some strains developing resistance to current front-line anti-malarials. Plasmodium falciparum M1 alanyl aminopeptidase (PFM1AAP) is involved in the terminal stages of haemoglobin degradation and the generation of an amino acid pool to support parasitic growth and development. This represents a promising new anti-malarial target where inhibition of this enzyme is lethal to the malarial parasite. To start understanding ligand recognition and binding in PFM1AAP for future drug design efforts, we explored (un)binding pathways of substrates, inhibitors and a product in the enzyme using steered molecular dynamics (sMD) simulations. The results of our simulations further clarify the substrate entrance and exit pathways to the buried active site of PFM1AAP and identify their respective physiochemical profiles. Our SMD simulations reveal several binding characteristics: (1) A substrate/inhibitor recognition mechanism, (2) active migration into the entrance channel, (3) a water reservoir which facilitates correct substrate orientation, (4) a molecular gate controlling product egress. Furthermore, several critical residues have been identified that in concert, facilitate these processes. These residues are now the focus of a mutagenesis study to fortify the in silico predictions. We observe differences in (un)binding pathways of substrates, inhibitors and product. A novel residue-based network analysis has been developed to highlight repetitive rather than sporadic events in the multiple sMD simulation trajectories of ligand (un)binding. Our work paves the way toward designing novel potent PFM1AAP inhibitors for the treatment of malaria.

Sitagliptin does not inhibit the M1 alanyl aminopeptidase from Plasmodium falciparum.

The M1 alanyl aminopeptidase from Plasmodium falciparum has been shown to be an essential hemoglobinase enzyme, catalyzing the final stages of hemoglobin break-down within intra-erythrocytic parasites 1. Recently there has been much interest in this protease as a potential drug target for the development of novel antimalarials [1–13]. In a recent report, Krishnamoorthy and Achary propose that Sitagliptin may serve as a potent competitive inhibitor of the M1 alanyl aminopeptidase enzyme from Plasmodium falciparum [14]. The molecule of interest, the M1 alanine aminopeptidase or PfA-M1, has been well studied and characterized by our group and others (For examples of recent papers, see [1–13]). To date, multiple published X-ray crystal structures and models are available of inhibitors bound to the active site of PfA-M1. Krishnamoorthy and Achary report a docking analysis of the enzyme with “about 100 low molecular weight protease inhibitors …”. The docking results were validated by inclusion of specific substrate (Ala-β-naphthylamide) from which they calculated an in silico Km value that was closely correlated with experimental data. Unfortunately, this correlation with experimental data was not observed with the selected positive inhibitor control, Bestatin. The interaction / inhibition of PfAM1 by Bestatin has been reported previously [1, 9, 12, 15] and the dipeptide analog has an in vitro Ki in the nM range for PfAM1. The in silico value calculated was ~ 100 μM, indicating that the parameters defined for docking were likely inadequate for the cation-occupied active site. The article states that Sitagliptin is the most potent in silico inhibitor with a Ki(avg) of 2 uM (however only 8/100 docking results were provided in Supplementary Table 1). We have completed an in vitro analysis of the effect of Sitagliptin on the activity of both PfA-M1 and the second neutral aminopeptidase, PfA-M17. Aminopeptidase activity assays were carried out in 200 µl total volume in 50 mM Tris-HCl pH 8.0, at 37 °C (with the addition of 2 mM CoCl2 for PfA-M17). Following a 10 min incubation of enzyme and Sitagliptin (0 – 0.5 mM), reactions were initiated by addition of fluorigenic substrate (L-Leucine-7-amido-4-methylcoumarin). Progress curves were monitored using a spectrofluorimeter until a final steady-state velocity was reached. We determined the inhibitory kinetics via Ki values from Dixon plots of 1/υs versus inhibitor concentration when [S]<<KM. We report here that this compound had no effect on the aminopeptidase activity of either enzyme. in silico drug docking suffers from several problems, including modeling the physics of the system, solvent effects, dynamics, and the difficulty in accurately ranking the docked results, and therefore relies critically on validation by experiment. It is vital that we rigorously test hypotheses generated from webservers to ensure that these algorithms continue to improve in their accuracy and hence usefulness. Blanket statements about efficacy from untested in silico studies will confound the literature and waste precious resources by following up on false positives identified in poorly controlled in silico studies. Computational biology has an important role to play in research and drug design; however, it is absolutely vital that we apply critical evaluation of results obtained to ensure that it becomes a robust method in the future.

Anti-malaria drug development targeting the M1 alanyl and M17 leucyl aminopeptidases

The M1 alanyl aminopeptidase and M17 leucyl aminopeptidase are critical to the growth and development of malaria parasites inside host erythrocytes. Potent aminopeptidase inhibitors kill malaria parasites in culture and are also active in vivo against murine malaria. Functional recombinant enzyme studies have been used to decipher the three-dimensional structures of both enzymes that together with new and specific inhibitors are facilitating structure-activity- relationship (SAR) and functional studies. Here we review the progress made in our knowledge of these two enzymes which is bringing them closer to being validated anti-malarial drug targets.

The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations

Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1–3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 Å-long channel and at the opening of the 30 Å-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.