The Most Potent Organophosphorus Inhibitors of Leucine Aminopeptidase. Structure-Based Design, Chemistry, and Activity

Abstract

A new class of very potent inhibitors of cytosol leucine aminopeptidase (LAP), a member of the metalloprotease family, is described. The X-ray structure of bovine lens leucine aminopeptidase complexed with the phosphonic acid analogue of leucine (LeuP) was used for structure-based design of novel LAP inhibitors and for the analysis of their interactions with the enzyme binding site. The inhibitors were designed by modification of phosphonic group in the LeuP structure toward finding the substituents bound at the S' side of the enzyme. This resulted in two classes of compounds, the phosphonamidate and phosphinate dipeptide analogues, which were synthesized and evaluated as inhibitors of the enzyme. The in vitro kinetic studies for the phosphinate dipeptide analogues revealed that these compounds belong to the group of the most effective LAP inhibitors found so far. Their further modification at the P1 position resulted in more active inhibitors, hPheP[CH(2)]Phe and hPheP[CH(2)]Tyr (K(i) values 66 nM and 67 nM, respectively, for the mixture of four diastereomers). The binding affinities of these inhibitors toward the enzyme are the highest, if considering all compounds containing a phosphorus atom that mimic the transition state of the reaction catalyzed by LAP. To evaluate selectivity of the designed LAP inhibitors, additional tests toward aminopeptidase N (APN) were performed. The key feature, which determines their selectivity, is structure at the P1' position. Aromatic and aliphatic substituents placed at this position strongly interact with the LAP S1' binding pocket, while a significant increase in binding affinity toward APN was observed for compounds containing aromatic versus leucine side chains at the P1' position. The most selective inhibitor, hPheP[CH(2)]Leu, binds to LAP with 15 times higher affinity than to APN. One of the studied compounds, hPheP[CH(2)]Tyr, appeared to be very potent inhibitor of APN (K(i) = 36 nM for the mixture of four diastereomers). The most promising LAP inhibitors designed by computer-aided approach, the phosphonamidate dipeptide analogues, were unstable at pH below 12, because of the P-N bond decomposition, which excluded the possibility of determination of their binding affinities toward LAP.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

“…At the entrance, a helix (␣14) with a 90°bend confines the pore size to Ϸ13-Å diameter. More recently, a new class of metallo-aminopeptidase inhibitors, phosphinate dipeptide analogues, were designed by using a computer-aided approach by Grembecka et al (15); 1 of these hPheP[CH 2 ]Phe, Co4, exhibited more potent inhibition of rPfA-M1 (K i ϭ 79 nM) than bestatin. Both bestatin and Co4 had similar killing activity against malaria parasites in culture; IC 50 bestatin range 8-14 M and Co4 is 24-62 M (Fig.…”

“…We have demonstrated that the aminopeptidase inhibitor bestatin, an antibiotic and natural analogue of the dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents P. falciparum malaria growth in culture (13,14). More recently, it was shown not only that synthetic phosphinate dipeptide analogues that inhibit metallo-aminopeptidases prevented the growth of wildtype and the chloroquine-resistant parasites in culture but also that one compound, hPheP[CH 2 ]Phe (termed compound 4, Co4), reduced a murine infection of Plasmodium chabaudi chabaudi by 92% compared with controls (15,16). Importantly, Co4 was found to cause no toxicity in these in vivo studies (16).…”

Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions

“…The fluoro-substituents sit deeper into the S1 pocket than any other hydroxamic-acid based inhibitor. In fact, the only other PfA-M1 inhibitor that has probed this region is the organophosphorus aminopeptidase inhibitor, Co4 [9,10,23]. Although the fluoro groups of 10o did not displace any of the ordered water molecules as anticipated from the 9f:PfA-M1 structure, they have entered into an intricate network of watermediated hydrogen-bonds, in which the fluorine atoms act as acceptors.…”

Stereoselective Synthesis of α-Amino-C-phosphinic Acids and Derivatives

“…Finally, hydrolysis of compounds (R)-46 with 6 N HCl at reflux followed by treatment with propylene oxide, provided the α-amino-C-phosphinic acid (R)-45 in 84% yield (Scheme 24). Taking into account that phosphinic dipeptides, although tested as stereoisomeric mixtures, are considered amongst the most potent inhibitors of bizinc cytosolic leucine aminopeptidase (LAP) [62], Mucha, et al [63] carried out the chiral HPLC separation of all stereoisomers of phosphinic dipeptide homophenylalanyl-phenylalanine derivative 40 and 41 on a quinidine carbamate modified silica stationary phase 42 (Scheme 23). Significant differences in inhibition of the unprotected derivatives 43 and 44 show the importance of the molecular stereochemistry in spatial interaction with the enzyme active site.…”