ABSTRACT
Plasmepsin 4 (PM4) is a digestive vacuole enzyme found in all Plasmodium species examined to date. While P. falciparum has three additional aspartic proteinases in its digestive vacuole in addition to plasmepsin 4, other Plasmodium species have only PM4 in their digestive vacuole. Therefore, PM4 may be a good target for the development of an antimalarial drug. This study presents data obtained with PM4s from several Plasmodium species. Low nanomolar K(i) values have been observed for all PM4s studied.
Subject(s)
Antimalarials/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Plasmodium/enzymology , Protease Inhibitors/pharmacology , Amino Acids/metabolism , Amino Acids/pharmacology , Animals , Antimalarials/metabolism , Aspartic Acid Endopeptidases/isolation & purification , Aspartic Acid Endopeptidases/metabolism , Chromatography, Affinity , Chromatography, Gel , Enzyme Precursors/metabolism , Gene Expression , Inclusion Bodies , Models, Molecular , Plasmodium/genetics , Primaquine/metabolism , Primaquine/pharmacology , Protease Inhibitors/metabolism , Protein Renaturation , Protozoan Proteins/isolation & purification , Vacuoles/enzymologyABSTRACT
Plasmepsin IV (Plm IV) is one of the aspartic proteases present in the food vacuole of the malaria parasite Plasmodium falciparum involved in host hemoglobin degradation by the parasite. Using a series of previously synthesized plasmepsin inhibitors [Ersmark, K., et al. (2005) J. Med. Chem. 48, 6090-106], we report here experimental data and theoretical analysis of their inhibitory activity toward Plm IV. All compounds share a 1,2-dihydroxyethylene unit as the transition state mimic. They possess symmetric P1 and P1' side chains and either a diacylhydrazine, a five-membered oxadiazole ring, or a retroamide at the P2 and P2' positions. Experimental binding affinities are compared to those predicted by the linear interaction energy (LIE) method and an empirical scoring function, using both a crystal structure and a homology model for the enzyme. Molecular dynamics (MD) simulations of the modeled complexes allow a rational interpretation of the structural determinants for inhibitor binding. A ligand bearing a P2 and P2' symmetric oxadiazole which is devoid of amide bonds is identified both experimentally and theoretically as the most potent inhibitor of Plm IV. For the P2 and P2' asymmetric compounds, the results are consistent with earlier predictions regarding the mode of binding of this class of inhibitors to Plm II. Theoretical estimation of selectivity for some compounds is also reported. Significant features of the Plm IV binding pocket are discussed in comparison to related enzymes, and the results obtained here should be helpful for further optimization of inhibitors.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Plasmodium falciparum/enzymology , Protease Inhibitors , Animals , Binding Sites , Computer Simulation , Crystallography, X-Ray , Models, Chemical , Models, Molecular , Molecular Conformation , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Protein Binding , Structure-Activity RelationshipABSTRACT
A series of HIV-1 protease mutants has been designed in an effort to analyze the contribution to drug resistance provided by natural polymorphisms as well as therapy-selective (active and non-active site) mutations in the HIV-1 CRF_01 A/E (AE) protease when compared to that of the subtype B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pretherapy B and AE proteases. Inhibition analysis with ritonavir, indinavir, nelfinavir, amprenavir, saquinavir, lopinavir, and atazanavir revealed that the natural polymorphisms found in A/E can influence inhibitor resistance. It was also apparent that a high level of resistance in the A/E protease, as with B protease, is due to it aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pretherapy B protease showed that the ability of atazanavir to maintain its binding affinity for variants containing some resistance mutations is due to its unique interactions with flap residues. This structure also explains why the I50L and I84V mutations are important in decreasing the binding affinity of atazanavir.
Subject(s)
Drug Resistance, Viral/genetics , HIV Protease Inhibitors/chemistry , HIV Protease/genetics , Oligopeptides/pharmacology , Pyridines/pharmacology , Amino Acid Substitution , Atazanavir Sulfate , Binding Sites , Crystallography, X-Ray , HIV Protease/chemistry , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Polymorphism, Genetic , Structure-Activity RelationshipABSTRACT
The first macrocyclic inhibitor of the Plasmodium falciparum aspartic proteases plasmepsin I, II, and IV with considerable selectivity over the human aspartic protease cathepsin D has been identified. A series of macrocyclic compounds were designed and synthesized. Cyclizations were accomplished using ring-closing metathesis with the second generation Grubbs catalyst. These compounds contain either a 13-membered or a 16-membered macrocycle and incorporate a 1,2-dihydroxyethylene as transition state mimicking unit. The binding mode of this new class of compounds was predicted with automated docking and molecular dynamics simulations, with an estimation of the binding affinities through the linear interaction energy (LIE) method.
Subject(s)
Aspartic Acid Endopeptidases/antagonists & inhibitors , Macrocyclic Compounds/pharmacology , Plasmodium falciparum/enzymology , Protease Inhibitors/pharmacology , Animals , Binding Sites , Binding, Competitive , Cathepsin D/antagonists & inhibitors , Crystallography, X-Ray , Cyclization , Drug Design , Humans , Macrocyclic Compounds/chemical synthesis , Macrocyclic Compounds/chemistry , Models, Chemical , Models, Molecular , Molecular Conformation , Plasmodium falciparum/drug effects , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Protozoan Proteins , Structure-Activity RelationshipABSTRACT
A series of inhibitors of the malarial aspartic proteases Plm I and II have been synthesized with L-mannitol as precursor. These inhibitors are characterized by either a diacylhydrazine or a five-membered oxadiazole ring replacing backbone amide functionalities. Molecular dynamics simulations were applied in the design process. The computationally predicted Plm II Ki values were generally in excellent agreement with the biological results. The diacylhydrazine was found to be superior over the oxadiazole as an amide bond replacement in the Plm I and II inhibitors studied. An extensive flexibility of the S2' pocket was captured by the simulations predicting the binding mode of the unsymmetrical inhibitors. Plm I and II inhibitors with single digit nanomolar Ki values devoid of inhibitory activity toward human Cat D were identified. One compound, lacking amide bonds, was found to be Plm IV selective and very potent, with a Ki value of 35 nM.
