Macrocycles mimic the extended peptide conformation recognized by aspartic, serine, cysteine and metallo proteases

It has been previously demonstrated that aspartic, serine, metallo and cysteine proteases bind to their inhibitors and substrate analogues in a single conformation, the saw-tooth or extended beta-strand. Consequently a generic approach to the development of protease inhibitors is the use of constraints that conformationally restrict putative inhibitor molecules to an extended form. In this way the inhibitor is pre-organized for binding to a protease and does not need to rearrange its structure. One constraining device that has proven to be effective for such pre-organization is macrocyclization. This article illustrates the general principle that macrocycles, especially those composed of 3-4 amino acids and usually 13-17 ring atoms, can effectively mimic the extended conformation of short peptide sequences. Such structure-stabilising macrocycles are stable to degradation by proteases, valuable components of potent protease inhibitors, and in many cases they are also bioavailable.     

Countering cooperative effects in protease inhibitors using constrained beta-strand-mimicking templates in focused combinatorial libraries

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.     

Structure-based design: synthesis and biological evaluation of a series of novel cycloamide-derived HIV-1 protease inhibitors

The structure-based design and synthesis of a series of novel nonpeptide HIV protease inhibitors are described. The inhibitors were designed based upon the X-ray crystal structure of inhibitor 1 (UIC-94017)-bound HIV-1 protease. The inhibitors incorporated 3-hydroxysalicyclic acid-derived acyclic and cyclic P2 ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. The inhibitors contain only two chiral centers and are readily synthesized in optically active form utilizing Sharpless asymmetric epoxidation, regioselective epoxide opening, and ring-closing olefin metathesis using Grubbs' catalyst as the key steps. We have synthesized 13-15-membered cycloamides and evaluated their HIV-1 protease enzyme inhibitory and antiviral activities in MT-2 cells. Interestingly, all cycloamide-derived inhibitors are noticeably more potent than the corresponding acyclic compounds. The ring size and substituent effects were investigated. It turned out that the 14-membered saturated ring is preferred by the S(1)-S(2) active sites of HIV-1 protease. Macrocycle 26 showed excellent enzyme inhibitory potency with a K(i) value of 0.7 nM and an antiviral IC(50) value of 0.3 microM. In view of their structural simplicity and preliminary interesting results, further optimization of these inhibitors is underway.     

Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.     

Conformational selection of inhibitors and substrates by proteolytic enzymes: implications for drug design and polypeptide processing

Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.     

Microwave-accelerated synthesis of P1'-extended HIV-1 protease inhibitors encompassing a tertiary alcohol in the transition-state mimicking scaffold

Two series of P1'-extended HIV-1 protease inhibitors comprising a tertiary alcohol in the transition-state mimic exhibiting Ki values ranging from 2.1 to 93 nM have been synthesized. Microwave-accelerated palladium-catalyzed cross-couplings were utilized to rapidly optimize the P1' side chain. High cellular antiviral potencies were encountered when the P1' benzyl group was elongated with a 3- or 4-pyridyl substituent (EC50 = 0.18-0.22 microM). X-ray crystallographic data were obtained for three inhibitors cocrystallized with the enzyme.     

Design and synthesis of potent C(2)-symmetric diol-based HIV-1 protease inhibitors: effects of fluoro substitution.

Implementation of derivatized carbohydrates as C(2)-symmetric HIV-1 protease inhibitors has previously been reported. With the objective of improving the anti-HIV activity of such compounds, we synthesized a series of fluoro substituted P1/P1' analogues. These compounds were evaluated for antiviral activity toward both wild type and mutant virus. The potency of the analogues in blocking HIV-1 protease was moderate, with K(i) values ranging from 1 to 7 nM. Nonetheless, compared to the parent nonfluorous inhibitors, a majority of the compounds exhibited improved antiviral activity, for example the 3-fluorobenzyl derivative 9b, which had a K(i) value of 7.13 nM and displayed one of the most powerful antiviral activities in the cellular assay of the series. Our results strongly suggest that fluoro substitution can substantially improve antiviral activity. The X-ray crystal structures of two of the fluoro substituted inhibitors (9a and 9f) cocrystallized with HIV-1 protease are discussed.     

Development of conformationally restricted analogues of bradykinin and somatostatin using constrained amino acids and different types of cyclization

The structure-based design of peptide drugs requires the knowledge of the bioactive conformation. Studies on this receptor-bound 3D structure require linear or cyclic analogues with strongly reduced flexibility, but high biological activity, since only analogues with retained potency have preserved the bioactive conformation. Constrained amino acids containing double bonds or bulky substituents at the N(alpha)-, C(alpha)- and C(beta)-atom as well as at the aromatic ring atom were successfully applied to obtain potent and stable analogues of bradykinin and somatostatin, which due to their restricted conformation were suitable objects for conformational studies. Besides the generation of constrained cyclic analogues with improved biological and pharmacological properties, cyclic peptides were used as convenient models for the study of turn formations. Cyclization of the linear peptide bradykinin was performed by linking the N-terminus and the C-terminus, and in both bradykinin and somatostatin by cyclization using the amino acid side chains and by backbone cyclization. The later requires the introduction of N(alpha)-functionalised amino acids for ring closure which can be performed either through incorporation of N(alpha)-functionalised amino acids or dipeptide building units. Conformational analysis of a cyclic bradykinin analogue by means of NMR-studies together with molecular dynamics simulation led to a quasicyclic 3D structure with two turns and together with other 3D structures provided a pharmacophore model of bradykinin antagonists.     

Design and synthesis of HIV-1 protease inhibitors incorporating oxazolidinones as P2/P2' ligands in pseudosymmetric dipeptide isosteres

A series of novel HIV-1 protease inhibitors based on two pseudosymmetric dipeptide isosteres have been synthesized and evaluated. The inhibitors were designed by incorporating N-phenyloxazolidinone-5-carboxamides into the hydroxyethylene and (hydroxyethyl)hydrazine dipeptide isosteres as P2 and P2' ligands. Compounds with (S)-phenyloxazolidinones attached at a position proximal to the central hydroxyl group showed low nM inhibitory activities against wild-type HIV-1 protease. Selected compounds were further evaluated for their inhibitory activities against a panel of multidrug-resistant protease variants and for their antiviral potencies in MT-4 cells. The crystal structures of lopinavir (LPV) and two new inhibitors containing phenyloxazolidinone-based ligands in complex with wild-type HIV-1 protease have been determined. A comparison of the inhibitor-protease structures with the LPV-protease structure provides valuable insight into the binding mode of the new inhibitors to the protease enzyme. Based on the crystal structures and knowledge of structure-activity relationships, new inhibitors can be designed with enhanced enzyme inhibitory and antiviral potencies.     

5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: potent nonpeptidic inhibitors of HIV protease

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S(3)' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S(3)' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.     

Renin inhibitors containing conformationally restricted P1-P1' dipeptide mimetics

A series of renin inhibitors containing lactam-bridged P1-P1' dipeptide mimetics based on the ACHPA (4(S)-amino-5-cyclohexyl-3(S)-hydroxypentanoic acid) design was studied. The inhibitors were obtained by aldol addition of various lactams with N alpha-Boc-L-cyclohexylalaninal, followed by Boc group removal and acylation with Boc-Phe-His. The aldol diastereomer having the S configuration at the two newly generated stereogenic centers gave optimal enzyme inhibition. Potency was further enhanced in the gamma-lactam ring series by substitution with small hydrophobic groups to mimic the P1' side chain of the renin substrate. Thus, 2(S)-[(Boc-L-phenylalanyl-L-histidyl)amino]-3-cyclohexyl-1(S)-hydroxyl-1 - (1,5,5-trimethyl-2-oxopyrrolidin-3(S)-yl)propane (34) has an IC50 of 1.3 nM in the human plasma renin assay. A variety of substituents on the lactam nitrogen are tolerated and can be used to vary the physical properties of the inhibitor. By using a model of the human renin active site, the conformation of 34 in the enzyme-inhibitor complex is proposed. This modeled conformation is very similar to the solid-state conformation of 2(S)-[(Boc-L-phenylalanyl-L-histidyl)amino]-3-cyclohexyl-1(S)-hydroxyl- 1-(1-methyl-2-oxopyrrolidin-3(S)-yl)propane (36), the structure of which was determined by single-crystal X-ray diffraction analysis. The most potent ACH-PA-lactam renin inhibitors show good selectivity when assayed against other types of aspartic proteinases. By varying the lactam ring substituents, potent and selective inhibitors of cathepsin D and cathepsin E can be obtained.     

Two-carbon-elongated HIV-1 protease inhibitors with a tertiary-alcohol-containing transition-state mimic

A new generation of HIV-1 protease inhibitors encompassing a tertiary-alcohol-based transition-state mimic has been developed. By elongation of the core structure of recently reported inhibitors with two carbon atoms and by varying the P1' group of the compounds, efficient inhibitors were obtained with Ki down to 2.3 nM and EC50 down to 0.17 microM. Two inhibitor-enzyme X-ray structures are reported.     

Design and fast synthesis of C-terminal duplicated potent C(2)-symmetric P1/P1'-modified HIV-1 protease inhibitors.

An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C(2)-symmetric C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds. These were synthesized with the objective to identify suitable P1/P1' substituents to provide inhibitors with improved antiviral activity. Groups with various physical properties were attached to the para-positions of the P1/P1' benzyloxy groups in the parent inhibitor. A p-bromobenzyloxy compound, prepared in only three steps from commercially available starting materials, was utilized as a common precursor in all reactions. The subsequent coupling reactions were completed within a few minutes and relied on palladium catalysis and flash heating with microwave irradiation. All of the compounds synthesized exhibited good inhibitory potency in the protease assay, with K(i) values ranging from 0.09 to 3.8 nM. A 30-fold improvement of the antiviral effect in cell culture, compared to the parent compound, was achieved with four of the inhibitors. The differences in K(i) values were not correlated to the differences in antiviral effect, efficiency against mutant virus, or reduced potency in the presence of human serum. The poorest enzyme inhibitors in fact belong to the group with the best antiviral effect. The binding features of two structurally related inhibitors, cocrystallized with HIV-1 protease, are discussed with special emphasis on the interaction at the enzyme/water phase.     

Recent advances in the medicinal chemistry of alpha-aminoboronic acids, amine-carboxyboranes and their derivatives

This article describes recent developments in the synthesis and biological activity of alpha-aminoboronic acids, amine-carboxyboranes and their derivatives as potential therapeutic agents. alpha-Amino acid analogues are of considerable interest as inhibitors of enzymes involved in amino acid and peptide metabolism. In particular, alpha-amino alkylphosphonic acids and alpha-amino alkylboronic acids, in which the carboxyl group of amino acids is replaced by a phosphonic acid or boronic acid function, respectively, constitute a unique class of amino acid mimics from which a number of potent enzyme inhibitors have been synthesized. The inhibitory activity mainly stems from the fact that the tetrahedral phosphonic moiety or the tetrahedral adduct of electrophilic boronic acid is a good mimic of the putative tetrahedral transition state or intermediate encountered in the enzymatic hydrolysis or formation of peptides. Since the peptide hydrolysis and formation invariably involves the tetrahedral high energy species in the course of the reaction, these amino acid mimics serve as a general key element for inhibitors of a broad spectrum of proteases and peptide ligases. Serine protease inhibitors provide promising compounds having a P site binding moiety and a boronic acid chelating moiety. The compounds have been shown to have high inhibitory activity.     

Molecular strategies to design an escape-proof antiviral therapy

Two antiviral approaches against the human immunodeficiency virus type 1 (HIV-1) were presented at the Antivirals Congress in Amsterdam. The common theme among these two separate therapeutic research lines is the wish to develop a durable therapy that prevents viral escape. We will present a brief overview of these two research lines and focus on our efforts to design an escape-proof anti-HIV therapy. The first topic concerns the class of HIV-1 fusion inhibitors, including the prototype T20 peptide and the improved versions T1249 and T2635, which were all developed by Trimeris–Roche. The selection of T20-resistant HIV-1 strains is a fairly easy evolutionary process that requires a single amino acid substitution in the peptide binding site of the viral envelope glycoprotein (Env) target. The selection of T1249-resistant HIV-1 strains was shown to require a more dramatic amino acid substitution in the viral Env protein, in particular the introduction of charged amino acid residues that cause resistance by charge-repulsion of the antiviral peptide. The third generation peptide T2635 remains active against all these HIV-1 escape variants because the charged residues within this peptide are “masked” by an introduced intra-helical salt bridge. This charge masking concept could facilitate the future design of escape-proof antiviral peptides. The second topic concerns the mechanism of RNA interference (RNAi) that we are currently employing to develop an antiviral gene therapy. One can make human T cells resistant to HIV-1 infection by a stable RNAi-inducing gene transfer, but the virus escapes under therapeutic pressure of a single inhibitor. Several options for a combinatorial RNAi attack to prevent viral escape will be discussed. The simultaneous use of multiple RNAi inhibitors turns out to be the most effective and durable strategy.     

An ethylenamine inhibitor binds tightly to both wild type and mutant HIV-1 proteases. Structure and energy study

An X-ray structure (resolution 2.2 A) of mutant HIV-1 protease (A71V, V82T, I84V) complexed with a newly developed peptidomimetic inhibitor with an ethylenamine isostere Boc-Phe-Psi[CH(2)CH(2)NH]-Phe-Glu-Phe-NH(2), denoted as OE, is described and compared with the complex of wild-type HIV-1 protease with the same inhibitor (resolution 2.5 A). OE shows tight binding to the wild type (K(i) = 1.5 nM) as well as mutant (K(i) = 4.1 nM) protease. The hydrogen bonds formed, in the case of hydroxyethylamine inhibitors, by a hydroxyl group are, in the case of OE inhibitors, replaced by a bifurcated hydrogen bond from the isosteric NH group to both catalytic aspartates Asp 25 and Asp 125. The binding modes of OE inhibitor to the wild type and mutant protease are similar. However, in the mutant protease, weaker van der Waals interactions of the mutated residues Val 84 and Val 184 with OE were found. This lack of interaction energy is compensated by a new aromatic hydrogen bond between the phenyl ring of the inhibitor in position P1 and the mutated residue Thr 182. Energy analysis based on molecular mechanics has been performed to distinguish between the static and dynamic backgrounds of disorder observed at the mutation sites Thr 82, Val 84, Thr 182, and Val 184.