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S S Abdel-Meguid

Publications and source records attributed to S S Abdel-Meguid.

At least 19 recordsLinked to original sources
PubMed2002

Controlling apoptosis by inhibition of caspases.

The intracellular cysteine proteinases grouped under the common name of caspases are important participants in the process of programmed cell death called apoptosis. Of the nearly fourteen mammalian members discovered thus far caspase 1 or (interleukin 1beta converting enzyme; ICE), and possibly other related family members also serve as activator of cytokines. In general, caspases act on a number of cellular targets including other caspase family members leading ultimately to apopto4 4is through a highly integrated and regulated biological, biochemical and genetic mechanism. The proper execution of apoptosis is crucial during developmental stages and continues to be of critical importance for the well being of the mature organism. However, in a number of degenerative diseases the pathological states are characterized by an exacerbated loss of certain types of cells, cellular death that has morphological characteristics of apoptosis. Fortunately, it has been known for sometime that induced apoptosis that proceeds through the activation of caspases can be inhibited to rescue these cells and allow them to remain viable. This realization has attracted attention towards caspases as likely targets for pharmacological intervention, believing that inhibition of their enzymatic activity in the compromised cells will prevent the unwanted high rate of cellular death. Here we survey natural and synthetic inhibitors of caspases that have been reported to date, including some commonly used peptide inhibitors that serve as "tool reagents" in research and others that have been used to map inhibitor binding interaction in the active site.

Animals↗
PubMed2000

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor.

Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo beta-lactamases. Reported here are the crystal structures of the native IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4 -(phenylb utyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 A (native) and 2.0 A (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo beta-lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 A compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the beta-lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo beta-lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo beta-lactamase inhibition with utility for future drug design.

Binding Sites↗
PubMed2000

Characterization of the in vitro and in vivo activity of monoclonal antibodies to human IL-18.

IL-18 is a cytokine with potent IFN-gamma inducing activities as well as an important mediator of Th1 polarized immune responses. In this study we demonstrated that IL-18 induces the concentration-dependent production of the proinflammatory mediators IFN-gamma, IL-6, and GM-CSF, but not the anti-inflammatory cytokine, IL-10 from peripheral blood lymphocytes in the presence of mitogen. Three neutralizing IL-18 monoclonal antibodies (MAbs) were investigated, one of which (2C10) inhibited IL-18 bioactivity with an IC50 of 0.1 nM and had a K(D) of 3.9 x 10(-11) M. A NOD/SCID mouse model engrafted with human peripheral blood lymphocytes was developed to test the in vivo efficacy of this MAb. The IFN-gamma production induced by LPS administration was inhibited approximately 90% by prior dosing of MAb 2C10. The therapeutic utility of a high-affinity IL-18 MAb may be of benefit in Th1-driven autoimmune diseases such as rheumatoid arthritis and Crohn's Disease, where elevated levels of IL-18 have been observed.

Animals↗
PubMed1999

Crystal structure of beta-ketoacyl-acyl carrier protein synthase III. A key condensing enzyme in bacterial fatty acid biosynthesis.

Beta-ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys(112) proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His(244) and Asn(274). The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1999

Cooperative structural dynamics and a novel fidelity mechanism in histidyl-tRNA synthetases.

The crystal structure of the Staphylococcus aureus histidyl-tRNA synthetase apoprotein has been determined at 2.7 A resolution. Several important loops in the active site either become disordered or adopt very different conformations compared to their ligand-bound states. These include the histidine A motif (Arg257-Tyr262) that is essential for substrate recognition, a loop (Gly52-Lys62) that seems to control the communication between the histidine and ATP binding sites, the motif 2 loop (Glu114-Arg120) that binds ATP, and the insertion domain that is likely to bind tRNA. These ligand-induced structural changes are supported by fluorescence experiments, which also suggest highly cooperative dynamics. A dynamic and cooperative active site is most likely necessary for the proper functioning of the histidyl-tRNA synthetase, and suggests a novel mechanism for improving charging fidelity.

Amino Acid Sequence↗
PubMed1999

Potent dipeptidylketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor.

Cathepsin B↗
PubMed1999

Molecular basis for triclosan activity involves a flipping loop in the active site.

The crystal structure of the Escherichia coli enoyl reductase-NAD+-triclosan complex has been determined at 2.5 A resolution. The Ile192-Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure-based inhibitor design.

Amino Acid Sequence↗
PubMed1998

Use of papain as a model for the structure-based design of cathepsin K inhibitors: crystal structures of two papain-inhibitor complexes demonstrate binding to S'-subsites.

Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexes and the rational design of novel cathepsin K inhibitors. Unlike previously known crystal structures of papain-inhibitor complexes, our papain structures show ligand binding extending deep within the S'-subsites. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leucinal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, were refined to 2.2- and 2.5-A resolution with R-factors of 0.190 and 0. 217, respectively. The S'-subsite interactions with the inhibitors are dominated by an aromatic-aromatic stacking and an oxygen-aromatic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yielded a novel, symmetric inhibitor selective for cathepsin K. Simultaneous exploitation of both S- and S'-sites provides a general strategy for the design of cysteine protease inhibitors having high specificity to their target enzymes.

Binding Sites↗
PubMed1998

Structure-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.

Benzoates↗
PubMed1998

Structural role of the 30's loop in determining the ligand specificity of the human immunodeficiency virus protease.

The structural basis of ligand specificity in human immunodeficiency virus (HIV) protease has been investigated by determining the crystal structures of three chimeric HIV proteases complexed with SB203386, a tripeptide analogue inhibitor. The chimeras are constructed by substituting amino acid residues in the HIV type 1 (HIV-1) protease sequence with the corresponding residues from HIV type 2 (HIV-2) in the region spanning residues 31-37 and in the active site cavity. SB203386 is a potent inhibitor of HIV-1 protease (Ki = 18 nM) but has a decreased affinity for HIV-2 protease (Ki = 1280 nM). Crystallographic analysis reveals that substitution of residues 31-37 (30's loop) with those of HIV-2 protease renders the chimera similar to HIV-2 protease in both the inhibitor binding affinity and mode of binding (two inhibitor molecules per protease dimer). However, further substitution of active site residues 47 and 82 has a compensatory effect which restores the HIV-1-like inhibitor binding mode (one inhibitor molecule in the center of the protease active site) and partially restores the affinity. Comparison of the three chimeric protease structures with those of HIV-1 and SIV proteases complexed with the same inhibitor reveals structural changes in the flap regions and the 80's loops, as well as changes in the dimensions of the active site cavity. The study provides structural evidence of the role of the 30's loop in conferring inhibitor specificity in HIV proteases.

Amino Acid Sequence↗
PubMed1998

Site-directed mutagenesis probing the catalytic role of arginines 165 and 166 of human cytomegalovirus protease.

Human cytomegalovirus (CMV) is a member of the Herpesviridae family of viruses that also includes herpes simplex viruses (HSV-1 and HSV-2), varicella-zoster virus (VZV), human herpes virus-6, 7, and 8 (HHV-6, HHV-7, and HHV-8), and Epstein-Barr virus (EBV). Each member of this family encodes a serine protease that is a potential target for antiviral therapeutic intervention. We recently reported the crystal structure of CMV proteases [Qiu, X., Culp, J. S., DiLella, A. G., Hellmig, B., Hoog, S. S., Janson, C. A., Smith, W. W., and Abdel-Meguid, S. S. (1996) Nature 383, 275-279] and proposed that the highly conserved Arg165 and Arg166 residues are involved in stabilizing the oxyanion intermediate in human herpes protease catalyzed reactions through the backbone NH and side chain, respectively. In the current study, site-directed mutagenesis was carried out to probe the catalytic function of these two amino acid residues. Substitution of Arg166 with an alanine has led to ablation of enzymatic activity without detectable change in CMV protease conformation, supporting suggestions from the crystal structure that Arg166 side chain plays a major role in catalysis. The wild-type has a Km = 138 +/- 17 microM and kcat = 19.9 +/- 1.1 min-1, while R166A has only residual activity, with a kcat = 0.012 +/- 0.001 min-1 and an unaltered Km = 145 +/- 18 microM. In the crystal structure, the side chain of Arg166 was shown previously to hold a water molecule that can act as a hydrogen-bond donor to the oxyanion and was thus proposed to stabilize the oxyanion intermediate. However, kinetic characterization of the mutant R165A only reveals a 2.7-fold lower activity than wild-type, with a Km = 166 +/- 19 microM and a kcat = 7.4 +/- 0.4 min-1. These results confirm that Arg165 side chain is not involved in the stabilization of the oxyanion. It is likely that Arg165 only utilizes the backbone NH for catalysis as suggested by the crystal structure.

Alanine↗
PubMed1997

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Binding Sites↗
PubMed1997

Active site cavity of herpesvirus proteases revealed by the crystal structure of herpes simplex virus protease/inhibitor complex.

Human herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are responsible for herpes labialis (cold sores) and genital herpes, respectively. They encode a serine protease that is required for viral replication, and represent a viable target for therapeutic intervention. Here, we report the crystal structures of HSV-1 and HSV-2 proteases, the latter in the presence and absence of the covalently bound transition state analog inhibitor diisopropyl phosphate (DIP). The HSV-1 and HSV-2 protease structures show a fold that is neither like chymotrypsin nor like subtilisin, and has been seen only in the recently determined cytomegalovirus (CMV) and varicella-zoster virus (VZV) protease structures. HSV-1 and HSV-2 proteases share high sequence homology and have almost identical three-dimensional structures. However, structural differences are observed with the less homologous CMV protease, offering a structural basis for herpes virus protease ligand specificity. The bound inhibitor identifies the oxyanion hole of these enzymes and defines the active site cavity.

Binding Sites↗
PubMed1997

Crystal structure of varicella-zoster virus protease.

Varicella-zoster virus (VZV), an alpha-herpes virus, is the causative agent of chickenpox, shingles, and postherpetic neuralgia. The three-dimensional crystal structure of the serine protease from VZV has been determined at 3.0-A resolution. The VZV protease is essential for the life cycle of the virus and is a potential target for therapeutic intervention. The structure reveals an overall fold that is similar to that recently reported for the serine protease from cytomegalovirus (CMV), a herpes virus of the beta subfamily. The VZV protease structure provides further evidence to support the finding that herpes virus proteases have a fold and active site distinct from other serine proteases. The VZV protease catalytic triad consists of a serine and two histidines. The distal histidine is proposed to properly orient the proximal histidine. The identification of an alpha-helical segment in the VZV protease that was mostly disordered in the CMV protease provides a better definition of the postulated active site cavity and reveals an elastase-like S' region. Structural differences between the VZV and CMV proteases also suggest potential differences in their oligomerization states.

Amino Acid Sequence↗