Inhibitor Side Chain Research Articles

Probing the role of interfacial residues in a dimerization inhibitor of HIV-1 protease

The importance of each side chain of a cross-linked interfacial peptide inhibitor of HIV-1 protease was evaluated using an alanine scanning approach. Whereas the parent inhibitor has an IC 50 value of 350 nM, values for the mutations reported here range from 280 –9200 nM. The relative importance or each residue was thus assigned and correlated to the solvent accessible surface area (SASA) exposed upon mutation.

C3-Thia and C3-carba isosteres of a carbocyclic influenza neuraminidase inhibitor, (3R,4R,5S)-4-acetamido-5-amino-3-propoxyl-1-cyclohexene-1-carboxylic acid

The importance of the oxygen atom in the C3 ether side chain of a carbocyclic influenza neuraminidase inhibitor 3 was investigated by replacement of the C3 ether oxygen atom of 3 with either a sulfur atom (compound 4) or a carbon atom (compound 5). The regio- and stereospecific syntheses of both isoteres are described starting from (−)-quinic acid.

X-Ray Crystallography of Catechol O-Methyltransferase: Perspectives for Target-Based Drug Development

Catechol O-methyltransferase (COMT) plays a role in the metabolism of catecholamines and some other substances containing the catechol structure. COMT also inactivates L-dopa, which is used as a drug in the treatment of Parkinson's disease. COMT catalyzes the transfer of the methyl group from the coenzyme S-adenosyl-L-methionine (AdoMet) to one of the phenolic hydroxyl groups of a catechol or substituted catechols. The presence of magnesium ions is required for the catalysis. The reaction products are O-methylated catechol and S-adenosyl-L-homocysteine (AdoHcy). COMT occurs in two forms, soluble and membrane-bound, the latter having a 50 amino-acids-long membrane anchor region. The membrane-bound COMT has about 10 times lower Km value than soluble COMT, but at saturating substrate concentrations, the catalytic number of both enzyme forms is similar. At low concentrations, catecholamines are methylated more efficiently by membrane-bound COMT. The active site of COMT consists of the AdoMet-binding domain and the catalytic site. The catalytic site is formed by a few amino acids that are important for substrate binding and catalysis of the methylation reaction, and by the metal ion. The Mg 2+ bound to the enzyme makes the hydroxyl groups of the catechol substrate more easily ionizable. In a vicinity of one hydroxyl from the bound substrate, there is a lysine residue, which accepts the proton of that moiety, and subsequently the methyl group is transferred from the AdoMet to the hydroxyl. The shallow groove in the active site of COMT allows the long side chains of inhibitors to reach the surface of the enzyme. The best inhibitors of COMT are nitrocatechols. Ionized molecules penetrate poorly the blood brain barrier. In clinical trials it has been shown that the beneficial effect on the L-dopa metabolism in the treatment of Parkinson's disease is reached with a peripheral COMT inhibitor such as entacapone.

Crystal Structures of Catalytic Subunit of cAMP-dependent Protein Kinase in Complex with Isoquinolinesulfonyl Protein Kinase Inhibitors H7, H8, and H89: STRUCTURAL IMPLICATIONS FOR SELECTIVITY

The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 A) and with H89 (2.3 A) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties.

Clarification of substrate specificity of papain by crystal analyses of complexes with covalent-type inhibitors

In order to investigate the stereo specificity of papain S n subsites ( n = 1–4) at the atomic level, two kinds of covalent-type inhibitors were designed based on the previous results on papain-E-64 and papain-E-64-c interactions, and their complex crystals with papain were analyzed by X-ray diffraction. The results show that the hydrophobic regions consisting of Val-133, Val-157 and Asp-158 and of Tyr-61, Gly-66 and Tyr-67 residues interact with the hydrophobic P 2 and P 3 side chains of inhibitors, thus indicating the function of the former and latter binding pockets as S 2 and S 3 subsites, respectively. Furthermore, the X-ray analysis suggests that the papain has no definite S n subsite of n ≥ 4, and the S 3-P 3 hydrophobic interaction is significantly affected by the P n side chain ( n ≥ 4) of both the substrate and the inhibitor.

E-64 Analogs as Inhibitors of Cathepsin L and Cathepsin S: Importance of the S 2-P 2 Interactions for Potency and Selectivity 1

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl, 1) in which the amino acid (AA) had been systematically varied were tested as inhibitors of cathepsins L and S. These E-64 analogs were designed to investigate whether selectivity for cathepsin L or cathepsin S could be attained by varying the amino acid bound to the essential epoxide ring which induces inhibition by alkylating the active site thiol of the cysteine proteases. The results indicate that the specificity of these analogs does not parallel that observed for substrates. This is possibly due to the fact that the direction of the peptide portion of these analogs, which is the reverse of that found for the substrate-like chloromethyl ketones (if the situation is analogous to papain), leads to differences in the orientation of the inhibitor side chain when bound in the S 2subsite compared to substrates. The greatest selectivity was obtained with HO-Eps-Arg-OBzl which exhibited an 89-fold preference for cathepsin L over cathepsin S. A change from the L to the D stereochemistry for the phenylalanine analogs resulted in a 19-fold drop in k 2/K i for cathepsin L and a 14-fold drop for cathepsin S. Both E-64 and Cbz-Phe-Ala-CH 2Cl form two hydrogen bonds with Gly 66 in the active site of papain. With the benzyl esters (HO-Eps-AA-OBzl) one of these hydrogen bonds is necessarily absent. In order to evaluate the importance of this hydrogen bond, three benzyl amide derivatives (HO-Eps-AA-NHBzl, 2) were synthesized. In all cases the potency of the inhibitor was increased and indeed the HO-Eps-Phe-NHBzl analog is 64-fold more potent than the corresponding benzyl ester. For cathepsin L, there is also a 237-fold preference for L-Phe over D-Phe in the benzyl amide analog. In conclusion, although the information available from S 2-P 2 interactions with substrates cannot be used to enhance the selectivity of the E-64 I analogs in a rational manner, the hydrogen-bonding interaction between the amide proton of the benzyl amid group in HO-Eps-AA-NHBzl and the S 2 subsite for both cathepsins L and S contributes to increase the potency of these types of inhibitors.

Modifications in a flexible surface loop modulate the isozyme-specific properties of mammalian alkaline phosphatases.

We have analyzed to what extent the surface loop domain of alkaline phosphatases (APs) is responsible for isozyme-specific functional properties. Unique AatII and RsrII restriction sites were introduced by site-directed mutagenesis at identical positions in murine tissue-nonspecific AP (TNAP) and human placental AP (PLAP) cDNAs to allow the homologous exchange of the loop domain of the TNAP (T domain) and PLAP (P domain) isozymes and the generation of the reciprocally chimeric molecules PLAP-T and TNAP-P. The introduction of the T loop into PLAP reduced the heat stability of PLAP-T to almost that of TNAP. The domain substitution was accompanied by a conformational change that resulted in the loss of immune reactivity with four of 17 epitope-mapped anti-PLAP monoclonal antibodies. The T and P loops provided stabilization to the side chain of specific uncompetitive AP inhibitors. The introduction of the T domain also conferred collagen-binding properties to PLAP-T accounting for half of the binding affinity of TNAP for collagen, while not affecting PLAP binding to IgG. Our data indicate that the surface loop determines overall enzyme stability, differs conformationally in the various isozymes, and modulates catalytic parameters in the presence of protein ligands, thus, accounting in part for isozyme-specific protein interactions.

15 gag Proteinase of myeloblastosis-associated virus: Specificity studies with substrate-based inhibitors

The specificity of the proteinase of myeloblastosis-associated virus (MAV) was studied with (a) 21 substrate-based inhibitors, (b) 9 inhibitors with pseudopalindrome sequences, (c) 8 chimeric inhibitors, and (d) 3 compounds designed as human immunodeficiency virus 1 (HIV-1) proteinase inhibitors. The central inhibitory unit (transition state or cleaved bond analog) and the role of the inhibitor side chains from P4 to P4′ were investigated. MAV proteinase prefers an aromatic side chain in P1 and a small aliphatic nonpolar chain in P2 and P2′. Residues in P5 and P4 positions are outside of the short catalytic cleft of the enzyme, but still influence binding considerably. The data obtained provide evidence that the MAV proteinase has generally lower specificity and poorer binding than the HIV proteinase.

Synthesis and biological activity of new conformationally restricted analogues of pepstatin.

A new statine derivative, 3-hydroxy-4-amino-5-mercaptopentanoic acid; cysteinylstatine (CySta), was synthesized and used to prepare a series of conformationally restricted analogues of pepstatin (Iva-Val-Val-Sta-Ala-Sta) in which the conformational constraint was introduced via a bis-sulfide connecting the appropriately substituted residues in the P1 and the P3 inhibitor side chains. The precursor peptide, Iva-Cys-Val-CySta-Ala-Iaa, was synthesized and alkylated with a series of dibromoalkanes and alkenes to produce the cyclic structures. This strategy permitted the carbon atom spacing between the P1 and the P3 inhibitor side chains to be systematically varied so as to produce inhibitors with 15-, 16-, and 17-membered ring systems. Additional non-cyclic analogues were synthesized as controls by alkylating the bisthiol intermediates with methyl iodide. The inhibitory potency of the analogues were determined against porcine pepsin and penicillopepsin by using standard enzyme kinetic assays. The cyclic inhibitor were found to be potent inhibitors of both aspartic proteases; inhibitor that contained a trans-2-butene link between the two sulfur atoms was found to be the most potent inhibitor with a Ki less than 1 nM against pepsin and 3.94 nM against penicillopepsin. This series of compounds illustrates a new type of conformational restriction that can be used to probe for the bioactive conformation of peptides.

New chiral blocks for introducing the side chain of HMG-CoA reductase inhibitors

A couple of versatile building blocks ( 8 and 9) of the side chain portion found in many HMG-CoA reductase inhibitors have been prepared. The successful introduction of the side chain to an aromatic ring by 8 or 9 has been demonstrated.

Variation in angiotensin-converting enzyme (ACE) inhibitor affinity at two binding sites on rat pulmonary ACE: influence on bradykinin hydrolysis.

1. ACE from rat lung and testis was characterized by radioligand binding studies using [125I]-Ro 31-8472, the radioiodinated hydroxy derivative of the potent ACE inhibitor cilazaprilat. 2. Analysis of the displacement of [125I]-Ro 31-8472 from ACE by ACE inhibitors of different structure by the LIGAND program was best fitted by a two binding site model for lung ACE and a one binding site model for testis ACE. 3. There was marked variation in ACE inhibitor binding affinity at the two binding sites of lung ACE across the panel of ACE inhibitors studied (equilibrium dissociation constant; Kd; pmol/L) for site one vs site two: cilazaprilat 40 +/- 3 vs 430 +/- 92*; lisinopril 25 +/- 1 vs 848 +/- 107**; and quinaprilat 4 +/- 1 vs 1869 +/- 720; *P less than 0.05; **P less than 0.005, t-test, n = 3). Reduction in binding affinity at site two of lung ACE was related to an increase in ACE inhibitor side chain length or complexity of carboxyl terminal moiety. ACE inhibitor binding affinity at the testis ACE binding site resembled site one of lung ACE. 4. Inhibition of bradykinin hydrolysis by lung ACE in the presence of increasing concentrations of cilazaprilat or quinaprilat was similar (F = 0.64; P greater than 0.05), suggesting that bradykinin cleavage predominates at ACE active site one. 5. The differences in ACE inhibitor affinity at the two ACE active sites has implications in physiological substrate selectivity, and may influence the pharmacodynamic effects of different ACE inhibitors.

Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease

Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID 50 values ranging from 10 −2 to 10 −4 m for inhibition of hydrolysis of succinylAlaAlaAla p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID 50 of 175 μ m and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.

Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid.

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.