Structure-based Designing of Inhibitors against Plasmepsins from Plasmodium Falciparum

The Plasmodium falciparum comprising four vacuolar plasmepsins (PMs) i.e. PMI, PMII, PMIV, and HAP (histo-aspartic protease) which are involved in hemoglobin (Hb) catabolism as well as a non-vacuolar plasmepsin, PMV responsible for virulent proteins export, represent promising targets to combat antimalarial drug resistance. This study reports the first detailed structural analysis and molecular dynamics simulation of PMV as an apoenzyme and its complexes with the PEXEL substrate as well as with the saquinavir inhibitor. The PMV-PEXEL complexed structure shows that the unique positions of Glu179 and Gln222 in PMV provides specificity to the PEXEL substrate consisting arginine at P3 position. The structural analysis also reveals that the S4 binding pocket in PMV contains bulkier residues Ile94, Ala98, Phe370 and Tyr472, that does not allow binding of pepstatin A, a potent inhibitor of most pepsin-like aspartic proteases. Saquinavir shows the highest binding affinity with PMV among the screened inhibitors. The P2 site carrying a flexible group and P3 position that is occupied by a bulky hydrophobic group of the inhibitor is preferred in the PMV substrate binding pocket. Results from the present study will aid in the design of more potent inhibitors of PMV. Further, we have demonstrated for the first time the use of soluble recombinant PMII for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high resolution crystal structures of PMII–KNI inhibitor complexes reveal interesting features modulating their differential potency. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI- 10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising antimalarial drug candidates. Based on our structural data, the novel KNI derivatives have been designed that can be further developed for potential clinical use. We have explored another interesting aspect of antimalarial drug design where we used PMII as a model system to investigate the inhibitory values of six well known HIV-1 protease inhibitors (PIs). These inhibitors block the activity of PMII with the Ki values in the low micromolar range ranging from 0.3-2.4 µM among which ritonavir exhibits the highest inhibition constant of 0.3 µM followed by lopinavir, saquinavir, nelfinavir, and indinavir. To understand the molecular basis of inhibition of PMII by PIs, the crystal structure of PMII in complex with ritonavir was solved. In the complexed structure, we have monitored the flexibility in the terminal P3 group in the inhibitor that can occupy multiple pockets. These results suggest that HIV-1 PIs could be further developed into effective antimalarial drugs that would target multiple PMs.

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