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    Vincent Madison

    Retired, Structural Chemistry, Department Member
    A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are... more
    A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP’s. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.Synthesis and optimization of novel tartrate TACE inhibitors are described.
    The lambda-dynamics method was used to calculate the relative binding free energies of inhibitors to the hepatitis C virus (HCV) protease. A total of seven HCV protease p-side product inhibitors were used in this study. The inhibitors are... more
    The lambda-dynamics method was used to calculate the relative binding free energies of inhibitors to the hepatitis C virus (HCV) protease. A total of seven HCV protease p-side product inhibitors were used in this study. The inhibitors are 6-mer peptides spanning P6-P1 (Ac-Asp-d-Glu-Leu-Ile-Cha-P1-CO(2)H). For this protein, S1 is a major hydrophobic pocket for binding. Binding of various residues to this pocket was investigated through free energy simulations and experimental inhibition constants. Several 300 ps lambda-dynamics simulations in explicit solvent were performed. The relative binding free energy was estimated from these simulations. From a single simulation, the inhibitors can be correctly classified into highly potent and weakly potent groups. The multiple simulations give an accurate rank ordering of inhibitor potency; computed and experimental binding free energies agree with 0.6 kcal/mol for five of the seven inhibitors. In addition, free energy perturbation (FEP) cal...
    Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination... more
    Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper
    ... Joseph Michalewsky,? Allan Schwartz,$ Sally Weatherford,: Joseph Triscari,t and David Nelsont Roche Research Center, Nutley, New Jersey 07110 Received July 20, 1992 ... Chem. 1992, in prese. (18) Tilley, JW; Danho, W ...... more
    ... Joseph Michalewsky,? Allan Schwartz,$ Sally Weatherford,: Joseph Triscari,t and David Nelsont Roche Research Center, Nutley, New Jersey 07110 Received July 20, 1992 ... Chem. 1992, in prese. (18) Tilley, JW; Danho, W ... 1990,526,95-102. (35) S hidi , K.; Lin, CW; Kopecka, H ...
    The limited efficacy and considerable side effects of currently available therapies for the treatment of hepatitis C virus (HCV) infection have prompted significant efforts toward the development of safe and effective new therapeutics.... more
    The limited efficacy and considerable side effects of currently available therapies for the treatment of hepatitis C virus (HCV) infection have prompted significant efforts toward the development of safe and effective new therapeutics. The pentapeptide alpha-ketoamides of type 1 were weak HCV inhibitors with a binding constant, Ki, above 5 microM. We envisioned that cyclization of a P2 phenyl side chain to a P3 capping group could enhance binding through an interaction of the resulting macrocycle with the methyl group of Ala156 on the enzyme backbone. The macrocyclic dipeptide moiety would also decrease the peptidic nature of the inhibitors. The synthesis of macrocyclic HCV inhibitors started from m-tyrosine methyl ester. Two consecutive couplings, first, with Boc-cyclohexylglycine and, then, with hept-6-enoic acid, provided compound 6. The alkene was converted to an alcohol via hydroboration. The key macrocyclization of phenol alcohol 7 was achieved through a Mitsunobu reaction. Both 16- and 17-membered macrocycles (8 and 21) were prepared. After hydrolysis, the macrocyclic acids (15 and 22) were coupled to the right-hand tripeptide (14) to afford alpha-hydroxyamides, which upon Dess-Martin periodinane oxidation furnished the desired alpha-ketoamides. Esters, acids, and amides were incorporated at the C-terminal of these peptides. These inhibitors were tested in an HCV protease continuous assay. The binding constants (Ki) indicated that the 16-membered macrocyclic inhibitors (23 and 24) were less potent than the 17-membered analogues (16-19). It was also evident that C-terminal acids (i.e., 17) and amides (18 and 19) (Ki range: 0.16-0.31 microM) were much better inhibitors than tert-butyl esters (16 and 23). The X-ray crystal structure of compound 17 bound to the enzyme revealed that the macrocycle formed a "donut"-shaped ring around the methyl group of Ala156. P2' phenyl and P1 propyl groups wrapped around the Lys136 side chain, forming a "C"-shaped clamp. The 17-membered macrocyclic inhibitors 17-19 were significantly more potent than the acyclic pentapeptide 1.
    Hepatitis C virus (HCV) NS3, when bound to NS-4A cofactor, facilitates development of mature virons by catalyzing cleavage of a polyprotein to form functional and structural proteins of HCV. The enzyme has a shallow binding pocket at the... more
    Hepatitis C virus (HCV) NS3, when bound to NS-4A cofactor, facilitates development of mature virons by catalyzing cleavage of a polyprotein to form functional and structural proteins of HCV. The enzyme has a shallow binding pocket at the catalytic site, making development of inhibitors difficult. We have designed, preorganized, and depeptidized macrocyclic inhibitors from P(4) to P(2)' and optimized binding to 0.1 microM. The structure of an inhibitor bound to the enzyme was also solved.
    The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide... more
    The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.
    The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are alpha-ketoamide-type HCV serine protease inhibitors with modest... more
    The NS3 protease of hepatitis C virus (HCV) has emerged as one of the best characterized targets for next-generation HCV therapy. The tetrapeptide 1 and pentapeptide 2 are alpha-ketoamide-type HCV serine protease inhibitors with modest potency. We envisioned that the 1,2,3,4-tetrahydroisoquinoline-3-carboxylamide (Tic) moiety could be cyclized to the P3 capping group. The resulting macrocycle could enhance the binding through its extra contact with the Ala156 methyl group. Macrocyclization could also provide a less peptidic HCV inhibitor. Synthesis started from dipeptide 5, which was obtained via a coupling of two amino acid derivatives. The N-terminal was capped as hept-6-enoylamide to give 6. Hydroboration of the double bond afforded alcohol 7, the precursor to the macrocycle 8. The macrocyclization was achieved under Mitsunobu conditions (PPh(3), ADDP). The macrocyclic acid 9 was then combined with appropriate right-hand fragments 12, 14, or 16, which was prepared from common intermediate 11. Finally, oxidation of alpha-hydroxyamide provided target molecule alpha-ketoamides 17, 18, and 21. The C-terminal esters were then elaborated to carboxylic acids 19 and 20, and amides 20 and 23. The inhibitors 17-23 were tested in HCV NS3 protease continuous assay. Tripeptide 17 was more potent than the larger acyclic tetrapeptide 1. The tetrapeptides 18-20 were as active as 17. Most significantly, the pentapeptides (21-23) were much better inhibitors (K(i) = 0.015-0.26 microM). The carboxylic acid (22) and amide (23) were 57-80 times more potent than the acyclic analogue 2. The X-ray crystal structure of compound 23 bound to the protease revealed that the macrocycle adopted a donutlike conformation and had close contact with the Ala156 methyl group. The ketone carbonyl formed a reversible covalent bond with Ser139. The n-propyl of P1 novaline and the aromatic ring of P2' phenylglycine formed a C-shaped clamp around the Lys136 side chain.
    Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are... more
    Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or polyethylene glycol (PEG)-interferon-alpha alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.
    Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to... more
    Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.
    Removal of the N-terminal formyl group from newly synthesized proteins by the enzyme peptide deformylase (PDF) is essential for normal growth of bacteria but not higher organisms. Recently, PDF has been explored as a target for novel... more
    Removal of the N-terminal formyl group from newly synthesized proteins by the enzyme peptide deformylase (PDF) is essential for normal growth of bacteria but not higher organisms. Recently, PDF has been explored as a target for novel antibiotics. Screening a collection of natural products for antimicrobial activity identified actinonin and two matlystatin analogs as potent PDF inhibitors. A number of synthetic analogs of these natural products were prepared and their inhibitory potency determined. Previous work has shown that PDF is an iron metalloproteinase also containing a catalytic glutamic acid residue. Ligation of the ferrous cation is an essential feature of potent inhibitors. The structures of actinonin, a matlystatin analog and a synthetic inhibitor complexed with PDF were determined by crystallography. A quantum mechanics/molecular mechanics (QM/MM) method was used to reproduce the geometry of known complexes, to predict the protonation state in the active site and to predict the geometry of additional complexes. The requirement for protonation of the active site glutamate anion is an important factor in understanding the potency of inhibitors with acidic iron-ligating groups such as hydroxamate and carboxylate. Even though potent inhibitors of PDF have been discovered, their bacteriostatic mechanism of action and the rapid development of resistance in vitro may limit their potential as antibacterial drugs.
    A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray... more
    A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1'-S3' pocket.
    Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to... more
    Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors has led to an acetylene containing series that demonstrates sub-nanomolar potency (K(i)) as well as excellent activity in human whole blood. These studies led to the discovery of highly potent TACE inhibitors with good DMPK profiles.

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