Ketomethylene Isosteres Research Articles

Visualizing Tetrahedral Oxyanion Bound in HIV-1 Protease Using Neutrons: Implications for the Catalytic Mechanism and Drug Design

HIV-1 protease is indispensable for virus propagation and an important therapeutic target for antiviral inhibitors to treat AIDS. As such inhibitors are transition-state mimics, a detailed understanding of the enzyme mechanism is crucial for the development of better anti-HIV drugs. Here, we used room-temperature joint X-ray/neutron crystallography to directly visualize hydrogen atoms and map hydrogen bonding interactions in a protease complex with peptidomimetic inhibitor KVS-1 containing a reactive nonhydrolyzable ketomethylene isostere, which, upon reacting with the catalytic water molecule, is converted into a tetrahedral intermediate state, KVS-1TI. We unambiguously determined that the resulting tetrahedral intermediate is an oxyanion, rather than the gem-diol, and both catalytic aspartic acid residues are protonated. The oxyanion tetrahedral intermediate appears to be unstable, even though the negative charge on the oxyanion is delocalized through a strong n → π* hyperconjugative interaction into the nearby peptidic carbonyl group of the inhibitor. To better understand the influence of the ketomethylene isostere as a protease inhibitor, we have also examined the protease structure and binding affinity with keto-darunavir (keto-DRV), which similar to KVS-1 includes the ketomethylene isostere. We show that keto-DRV is a significantly less potent protease inhibitor than DRV. These findings shed light on the reaction mechanism of peptide hydrolysis catalyzed by HIV-1 protease and provide valuable insights into further improvements in the design of protease inhibitors.

Synthesis of a Ketomethylene Isostere of the Fibrillating Peptide SNNFGAILSS

The direct synthesis of a ketomethylene isostere of the fibril-forming decapeptide SNNFGAILSS is presented with the goal of understanding how small structural changes alter the ability of such peptides to recognize each other for beta-sheet formation. The key synthetic step relies on a SmI(2)-mediated coupling of a N-tetrapeptidyl oxazolidinone with a simple acrylate followed by deprotection of the carboxylic acid and a peptide coupling step with the pentapeptide H-AILSS-NH(2).

Reprint of “Crystal structure of chemically synthesized HIV-1 protease and a ketomethylene isostere inhibitor based on the p2/NC cleavage site” [Bioorg. Med. Chem. Lett. 18 (2008) 4554-4557

Here we report the X-ray structures of chemically synthesized HIV-1 protease and the inactive [D25N]HIV-1 protease complexed with the ketomethylene isostere inhibitor Ac–Thr–Ile–Nle ψ[CO–CH 2]Nle–Gln–Arg.amide at 1.4 and 1.8 Å resolution, respectively. In complex with the active enzyme, the keto-group was found to be converted into the hydrated gem-diol, while the structure of the complex with the inactive D25N enzyme revealed an intact keto-group. These data support the general acid–general base mechanism for HIV-1 protease catalysis.

Crystal structure of chemically synthesized HIV-1 protease and a ketomethylene isostere inhibitor based on the p2/NC cleavage site

Here we report the X-ray structures of chemically synthesized HIV-1 protease and the inactive [D25N]HIV-1 protease complexed with the ketomethylene isostere inhibitor Ac–Thr–Ile–Nle ψ[CO–CH 2]Nle–Gln–Arg.amide at 1.4 and 1.8 Å resolution, respectively. In complex with the active enzyme, the keto-group was found to be converted into the hydrated gem-diol, while the structure of the complex with the inactive D25N enzyme revealed an intact keto-group. These data support the general acid–general base mechanism for HIV-1 protease catalysis.

Stereocontrolled Formation of Ketomethylene Isosteres through Tandem Chain Extension Reactions

A zinc-mediated chain extension reaction is the key step in the preparation of gamma-keto amides derived from amino acids. The use of tandem reaction sequences, in which the intermediate zinc enolate is trapped with electrophilic reagents, results in the incorporation of alpha-substituents, which mimic the side chains found in natural amino acid systems. Use of the chiral amino acid L-proline as a stereo-directing element provides a diastereoselective route to various ketomethylene isosteres.

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide α,β-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC 50 > 100 μM), the ketomethylene isosteres and tripeptide α,β-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC 50 = 11–39 μM). The Phe-Phe dipeptide inhibitors 18a– e are designed on the basis of computer modeling of the enzyme–inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 μM is obtained by condensation of the Phe-Phe dipeptide α,β-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC 50 value of 0.18 μM.

Dipeptidomimetic ketomethylene isosteres as pro-moieties for drug transport via the human intestinal di-/tripeptide transporter hPEPT1: design, synthesis, stability, and biological investigations.

Five dipeptidomimetic-based model prodrugs containing ketomethylene amide bond replacements were synthesized from readily available alpha,beta-unsaturated gamma-ketoesters. The model drug (BnOH) was attached to the C-terminus or to one of the side chain positions of the dipeptidomimetic. The stability, the affinity for the di-/tripeptide transporter hPEPT1, and the transepithelial transport properties of the model prodrugs were investigated. ValPsi[COCH(2)]Asp(OBn) was the compound with highest chemical stability in buffers at pH 6.0 and 7.4, with half-lives of 190 and 43 h, respectively. All five compounds showed high affinity for hPEPT1 (K(i) values < 1 mM), and PhePsi[COCH(2)]Asp(OBn) and ValPsi[COCH(2)]Asp(OBn) had the highest affinities with K(i) values of 68 and 19 microM, respectively. An hPEPT1-mediated transport component was demonstrated for the transepithelial transport of three compounds, a finding that was corroborated by hPEPT1-mediated intracellular uptake. The results indicate that the stabilized Phe-Asp and Val-Asp derivatives are promising pro-moieties in a prodrug approach targeting hPEPT1.

Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations.

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.

Chain extension of amino acid skeletons: preparation of ketomethylene isosteres

Ketomethylene isosteric replacements for peptide bonds were generated through a zinc carbenoid-mediated chain extension reaction in which a variety of amino acid-derived β-keto esters are converted to γ-keto esters in a single step. The reaction tolerates a variety of protecting groups and amino acid side chains with no epimerization of the amino acid stereocenter.

Potent bivalent thrombin inhibitors: replacement of the scissile peptide bond at P(1)-P(1)' with arginyl ketomethylene isosteres.

We have designed highly potent synthetic bivalent thrombin inhibitors, which consist of an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The bivalent inhibitors bind to the active site and the fibrinogen recognition exosite simultaneously. As a result, the inhibitors showed much higher affinity for thrombin than the individual blocking segments. Various arginyl ketomethylene isosteres ArgPsi[CO-CH(2)-X]P(1)' were incorporated into the bivalent inhibitors as P(1)-P(1)' segment to eliminate the scissile bond. The P(1)' residue is a natural or unnatural amino acid; specifically, the incorporation of mercaptoacetic acid exhibited superiority in synthesis and affinity for thrombin. Inhibitor 16, (D-cyclohexylalanine)-Pro-ArgPsi[CO-CH(2)-S]Gly-(Gly)(4)-Asp-Tyr-G lu- Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-(D-Glu)-OH, showed the lowest K(i) value of 3.5 +/- 0.5 x 10(-13) M, which is comparable to that (K(i) = 2.3 x 10(-13) M) of recombinant hirudin. Consequently we successfully reduced the size of the inhibitor from approximately 7 kDa of recombinant hirudin to approximately 2 kDa without losing the affinity.

Synthesis, Biological Activity and Molecular Modelling of the 53-54 Ketomethylene Analogue ofHEL(52-61) . .

Abstract: The synthesis of the 53-54 ketomethylene isostere of HEL(52-61), an antigenic decapeptide, was realised by convergent Fmoc solid-phase peptide synthesis. In . addition to the target peptide, an unexpected hydroxysuccinyl by-product was recovered. The two ketomethylene epimers did not exhibit any stimulating or competitive activity. Molecular modelling studies of the analogues-MHC-II 1-Ak complex.suggested a departure from the canonical polyproline II conformation which could account for the absence of binding to 1- Ak molecule.

First application of the Dakin-West reaction to fmoc chemistry: Synthesis of the ketomethylene tripeptide fmoc- Nα-Asp( tBu)-(R,S Tyr( tBu) Ψ(CO-CH 2)Gly-OH

A practical synthesis of a tripeptide containing a ketomethylene isostere, suitably protected for introduction in Fmos SPPS, has been carried out for the first time in Fmoc chemistry by using a modified Dakin-West reaction.

Synthesis of Xaa-Gly-Xaa′ keto-methylene tri-peptide isosteres incorporating phenylalanine, tyrosine and valine units

The utility of amino-acid derived β-ketosulfones in the synthesis of keto-methylene tri-peptide isosteres Xaa-Gly-Xaa′, incorporating phenylalanine, tyrosine and valine units is reported. Both L,L and L,D forms of the tripeptide systems have been prepared using this methodology.

Thrombin Inhibitors Based on Ketone Derivatives of Arginine and Lysine

Much attention is currently focused on inhibitors of thrombin as potential anticoagulants. We have previously reported thrombin inhibitors based on fragments of fibrinogen containing a ketomethylene isostere at P1-P'1. We now expand on these early findings by reporting on tripeptide based inhibitors of thrombin containing arginine or lysine ketones at the C-terminus. A large variety of such ketones have been studied and compared in their ability to increase the thrombin time in human plasma. In the case of arginine or lysine ketones the order of activity (i.e. decreasing IC50 TT) was: alkyl ketones < beta-ketoesters < difluoro beta-ketoamides < alkyloxymethyl ketones < fluoroketones. Lysine analogues were generally found to be ca. ten-fold less active than their arginine counterparts. However, in the case of alpha-ketoesters the lysine derivatives were superior to all the types of arginine ketones studied (including the arginine alpha-keto ester derived thrombin inhibitor). A mechanistic explanation of the relative inactivity of the arginine alpha-keto ester derivative is proposed. All the highly electrophilic ketones were found to be slow-binding with thrombin.

Design and synthesis of thrombin inhibitors

As part of our programme directed at the development of enzyme inhibitors based on transition-state mimics, we discovered in the early 1980s that P3-P3′ fragments of human fibrinogen Aα, containing the ketomethylene isostere Arg-Ψ-[COCH2]Gly at P1-P1′, were potent inhibitors of thrombin. Such low-molecular-weight inhibitors are expected to be clinically useful as anticoagultant drugs. In our more recent investigations, the P1-P1′ moiety has been replaced with various arginine or lysine ketones. The resulting compounds showed the following order of thrombin inhibitory potency: α-ketoesters > fluoroketones >alkoxymethylketones > difluoro-β-ketoamides >β-ketoesters >alkyl ketones. In contrast to all other lysine/arginine pairs studied previously, the inhibitor based on a lysine α-ketoester proved superior to the corresponding arginine analogue. A possible explanation for this finding is discussed. All the highly electrophilic ketones (e.g., fluoroketones) were found to exhibit slow-binding kinetics with thrombin, which is likely to be a disadvantage in clinical use. Alkoxymethyl ketones were devoid of such behaviour and have been developed further to yield nanomolar inhibitors of low molecular weight and good selectivity for thrombin. One of these ketones was found to compare favourably with known thrombin inhibitors in anticoagulant assays. The synthesis of various types of inhibitor mentioned above is described, together with structure-activity correlations for inhibition of thrombin.

HIV-1 protease inhibitors: Ketomethylene isosteres with unusually high affinity compared with hydroxyethylene isostere analogs

HIV protease is a member of the aspartic proteinase family of proteolytic enzymes which include pepsin and renin. In contrast to the enhanced affinity seen with renin and pepsin upon conversion of the transition-state isostere, ketomethylene, to the hydroxyethylene, a set of HIV protease inhibitors showed a reduction in affinity. This implies that interactions with the active site of other segments of the inhibitor than those of the transition-state analog must predominate in the case of HIV protease, and that observations made on mammalian aspartic proteinases do not necessarily apply to viral aspartic proteinases.

Inhibition of aminopeptidases by peptides containing ketomethylene and hydroxyethylene amide bond replacements

Inhibitors of aminopeptidase enzymes have been prepared by the synthesis of peptide substrate analogues in which the scissile amide bond has been replaced with the hydrolytically stable ketomethylene (-COCH2-) and hydroxyethylene [-CH(OH)CH2-] functionalities. Two synthetic strategies were used to prepare the inhibitors, and the advantages and disadvantages of each are discussed. The synthesis of peptides that contain the hydroxyethylene isostere was complicated by competing lactone and lactam formation, and attempts to prepare free N-terminal dipeptide hydroxyethylene isostere derivatives were unsuccessful. All ketomethylene isosteres examined were weak inhibitors of both leucine aminopeptidase and aminopeptidase M. However, the ketomethylene inhibitor LysK(RS)Phe (58) (Ki = 4 nM) is a potent inhibitor comparable to the natural product, arphamenine A (ArgKPhe; Ki = 2.5 nM). Normal Michaelis-Menten kinetics for inhibition of membrane leucine aminopeptidase are observed in the absence of magnesium ion, but nonlinear kinetics were obtained in the presence of Mg2+.