Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

Abstract

Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009 Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1′ library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1′-Gln-(Xaa). In both cases, P1 and P1′ were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1′ residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatographymass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1′-Phe pools revealed the favored amino acids at the P3, P2, P2′, and P3′ positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

Analysis of Binding Interactions of Pepsin Inhibitor-3 To Mammalian and Malarial Aspartic Proteases

“…For the other malarial enzymes, the K i values for the F2A mutant were no more than 3-fold higher or lower than wild-type. Current structural and substrate studies are consistent with the results of the F2A mutant (35). The structure of the uncomplexed PfPM2 revealed that the active site of PfPM2 is larger than commonly assumed based on structures of the enzyme in complex with inhibitors (36).…”

Activation, Proteolytic Processing, and Peptide Specificity of Recombinant Cardosin A

“…The peptide specificity assay was described in more detail elsewhere (18). Briefly, an aliquot of each pool of the P1 and P1Ј libraries giving a total peptide concentration of ϳ100 M was used in a spectrophotometric assay.…”

Development of Plasmodium falciparum Protease Inhibitors in the Past Decade (2002–2012)

“…Like these HIV-1 aspartyl protease inhibitors, most Plm I and II inhibitors mimic the tetrahedral intermediate formed during the aspartyl protease catalysis. There are several transition state analogue cores used for the design of Plm inhibitors [35,[51][52][53][54][55][56][57][58][59][60][61][62][63][64][65], but the most important include the statine core [54,56,57,64], the reversed-statine core [53,54], or a hydroxyethylamine motif (Fig. 4) [56,61,62].…”

“…(4). Transition-state mimicking groups in peptidomimetic plasmepsin inhibitors: reduced amine [52,59], statine [54,56,57,64], hydroxypropylamine [51], reversed-statine [53,54], dihydroxyethylene (C-and N-duplicated) [35,55,63], norstatine [58,60,65], and hydroxyethylamine [56,61,62].…”