Nitro As Zinc Binding Group For Inhibitor Of Zinc Protease

Zinc proteases,which are differentiated from other proteases by having a catalytically essential zinc ion at the active site,constitute an important class of proteolytic enzymes.They are widely throughout found in a variety of tissues and play key roles in numerous physiological processes,ranging from pathogenic infections to high blood pressure,and even cancer.The most studied drug design targets are angiotensin-converting enzyme(ACE),matrix metalloproteases(MMPs), etc.Carboxypeptidase A(CPA) and Thermolysin(TLN) are a leading prototypical enzyme for zinc proteases.Development of design principles of inhibitors for CPA and TLN would bear special importance because they can be translated to zinc proteases of medicinal value,leading to the discovery of potential lead compounds for drug development.Inhibitor design for most metalloproteases therefore has focused on small inhibitors capable of interacting with the prime side pockets,a peptidomimetic backbone and incorporating a zinc-binding group(ZBG).The general strategy of incorporating a ZBG,such as a carboxylic acid, sulfhydryl,phosphinate,hydroxamate,or formylhydroxylamine,for the designed inhibitors has proven to be highly successful.The exploration and discovery of more potent and selective ZBGs is required.There are two paths to elucidate drug-metalloprotein interactions by using bioinorganic model compounds screen for ZBG or examination of novel ZBG for use in enzyme inhibitor.CPA and TLN were among the first proteins to have their structures unraveled by X-ray crystallography and they have been studied extensively by biochemical and structural means.Thus,both CPA and TLN serve as prototypes for zinc proteases.Being an isostere of the carboxylate,the nitro group also shows a variety of coordinative fashions to the metal ions.Compared to the wide applications of the carboxylate as the ZBG in the inhibition of zinc proteases,to the best of our knowledge,the utilization of a nitro group as the ZBG has been rarely explored,is not found in zinc proteases inhibitors.Nitro-based compounds were designed rationally as inhibitors for CPA. Because±-2-benzyl-3-nitropropanoic acid(BNP) inhibits CPA most potently,the two optically active forms of BNP to explore the stereochem'istry associated with the inhibition were then synthesized. Introduction of the nitro group was achieved by the reaction of halogen-NO₂ exchange using Amberlite IRA-900 resin in the nitrite form.BNP can be obtained through treatment of the ester with lithium iodide in refluxing anhydrous ethyl acetate.Inhibitory activities against CPA were evaluated and their inhibitory constant values in Dixon method using O-(trans-p-chlorocinnamoyl)-L-3-phenylacctate(Cl-CPL) as substrate.In CPA inhibitory assays L-BNP showed potent inhibitory activity with the K_i value of 0.15μM.Its enantiomer was shown to be much less potent(K_i= 68μM).As a contrast,2-nitro-3-phenylpropaonic acid(PNP), 4-methyl-2-(nitromethyl)pentanoic acid(MNP) and(2-nitromethyl)-4-pentenoic acid(NMP) were also designed and synthesized.It can be found that MNP inhibits the CPA-catalyzed hydrolysis more potently than NMP, which should be ascribed to the preference of CPA to the substrate having a large hydrophobic side chain.Introduction of an aromatic ring further augmented the binding affinity of the inhibitors to CPA,which may be resulted from the additional 'edge-to-face' interactions of the benzyl side chain of BNP with aromatic enzyme residues.To explore the binding mode of L-BNP at the active site of CPA, CPA-L-BNP crystals for X-ray diffraction were then obtained by soaking the enzyme crystals into the solution containing racemic BNP and determined at a resolution of 1.7(?)(PDB ID code:2RFH).Single crystals grew in the P2₁ space group with one molecule in the asymmetric unit.The final model including all residues of CPA and L-BNP was refined in the 20-1.70(?).The nitro group of L-BNP acts as an asymmetric bidentate ligand of the zinc ion in an O,O'-chelation mode(2.10 and 3.06(?)).the two oxygen atoms(03 and 04) of the nitro moiety in L-BNP are engaged in hydrogen bonding with one of the carboxylate hydrogen atoms in Glu-270 and one of the guanidinium hydrogen atoms in Arg-127 with the distances of 2.60 and 2.87(?),respectively.To evaluate the metal binding interactions using AutoDock program,the nitro group in the BNP do coordinate to the zinc ion at the active site of CPA in an O,O'-bidentate fashion,but the hydrogen bonds are missing. In spite of being not a tetrahedron species,the nitrite in CPA-L-BNP complex exhibited all above the structurally essential features of the transition state.In this sense,the nitro group should be reasonably regarded as a new ZBG,L-BNP as a pseudotransition-state analog inhibitor for CPA.A series of inhibitors that bear nitro moiety have been evaluated as inhibitors for thermolysin.Optically form of 2-benzyl-3-nitropro panoic acid methyl ester(BNPM) and 2-benzyl-3-nitropropionamide(BNPA), racemic 2-nitro-3-phenylpropionic acid methyl ester(NPPM) and 2-nitro-3-phenylpropionamide(NPPA) have been synthesized and evaluated as inhibitors for TLN.Inhibitory activities of the synthesized nitro compound derivatives for thermolysin were estimated at pH 7.5 by the Dixon method's using 2-Furylacryloyl-Gly-Leu-NH₂ as substrate.The most potent inhibitor is L-BNPA having the K_i value of 7.6μM,it binds TLN better than the existing potent inhibitors bearing reversed hydroxamate such as L-HCONOH-β-Leu-NH₂(K_i= 53μM).The molecular modeling study for TLN and L-BNPA complex suggested that the two oxygen atoms of the nitrite also bind the zinc ions in a O,O'-bidentate fashion.Amides are more potent than esters,suggesting that the hydrogen atom on the amide nitrogen is possibly involved in the formation of hydrogen bond with backbone peptide carbonyl oxygen atom.On the basis of these results it may be concluded that the nitro-bearing zinc protease inhibitors are a new type of pseudo-transition state analogue inhibitors,although the nitro group is not a tetrahedron species,it also showed the similar binding modes as transition-state analog.Nitro group is a valuable moiety that can be useful as active site zinc coordination functionality in designing inhibitors for CPA and TLN.The novel design protocol may potentially be employed in designing inhibitors effective against zinc proteases such as ACE and MMP,generating inhibitors of therapeutic applications.