Biomedical subjects

D Joseph-McCarthy

Publications and source records attributed to D Joseph-McCarthy.

13 recordsLinked to original sources

PubMed2005

Bicyclic and tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors.

A novel pyridothiophene inhibitor of PTP1B was discovered by rational screening of phosphotyrosine mimics at high micromolar concentrations. The potency of this lead compound has been improved significantly by medicinal chemistry guided by X-ray crystallography and molecular modeling. Excellent consistency has been observed between structure-activity relationships and structural information from PTP1B-inhibitor complexes.

Crystallography, X-Ray↗

PubMed2001

Use of MCSS to design small targeted libraries: application to picornavirus ligands.

Computational methods were used to design structure-based combinatorial libraries of antipicornaviral capsid-binding ligands. The multiple copy simultaneous search (MCSS) program was employed to calculate functionality maps for many diverse functional groups for both the poliovirus and rhinovirus capsid structures in the region of the known drug binding pocket. Based on the results of the MCSS calculations, small combinatorial libraries consisting of 10s or 100s of three-monomer compounds were designed and synthesized. Ligand binding was demonstrated by a noncell-based mass spectrometric assay, a functional immuno-precipitation assay, and crystallographic analysis of the complexes of the virus with two of the candidate ligands. The P1/Mahoney poliovirus strain was used in the experimental studies. A comparison showed that the MCSS calculations had correctly identified the observed binding site for all three monomer units in one ligand and for two out of three in the other ligand. The correct central monomer position in the second ligand was reproduced in calculations in which the several key residues lining the pocket were allowed to move. This study validates the computational methodology. It also illustrates that subtle changes in protein structure can lead to differences in docking results and points to the importance of including target flexibility, as well as ligand flexibility, in the design process.

Benzimidazoles↗

PubMed2001

A structurally biased combinatorial approach for discovering new anti-picornaviral compounds.

BACKGROUND: Picornaviruses comprise a family of small, non-enveloped RNA viruses. A common feature amongst many picornaviruses is a hydrophobic pocket in the core of VP1, one of the viral capsid proteins. The pocket is normally occupied by a mixture of unidentified, fatty acid-like moieties, which can be competed out by a family of capsid-binding, antiviral compounds. Many members of the Picornaviridae family are pathogenic to both humans and livestock, yet no adequate therapeutics exist despite over a decade's worth of research in the field. To address this challenge, we developed a strategy for rapid identification of capsid-binding anti-picornaviral ligands. The approach we took involved synthesizing structurally biased combinatorial libraries that had been targeted to the VP1 pocket of poliovirus and rhinovirus. The libraries are screened for candidate ligands with a high throughput mass spectrometry assay. RESULTS: Using the mass spectrometry assay, we were able to identify eight compounds from a targeted library of 75 compounds. The antiviral activity of these candidates was assessed by (i) measuring the effect on the kinetics of viral uncoating and (ii) the protective effect of each drug in traditional cell-based assays. All eight of the candidates exhibited antiviral activity, but three of them were particularly effective against poliovirus and rhinovirus. CONCLUSIONS: The results illustrate the utility of combining structure-based design with combinatorial chemistry. The success of our approach suggests that assessment of small, targeted libraries, which query specific chemical properties, may be the best strategy for surveying all of chemical space for ideal anti-picornaviral compounds.

Animals↗

PubMed2001

Functional group placement in protein binding sites: a comparison of GRID and MCSS.

One approach to combinatorial ligand design begins by determining optimal locations (i.e., local potential energy minima) for functional groups in the binding site of a target macromolecule. MCSS and GRID are two methods, based on significantly different algorithms, which are used for this purpose. A comparison of the two methods for the same functional groups is reported. Calculations were performed for nonpolar and polar functional groups in the internal hydrophobic pocket of the poliovirus capsid protein, and on the binding surface of the src SH3 domain. The two approaches are shown to agree qualitatively; i.e., the global characteristics of the functional group maps generated by MCSS and GRID are similar. However, there are significant differences in the relative interaction energies of the two sets of minima, a consequence of the different functional form used to evaluate polar interactions (electrostatics and hydrogen bonding) in the two methods. The single sphere representation used by GRID affords only positional information, supplemented by the identification of hydrogen bonding interactions. By contrast, the multi-atom representation of most MCSS groups yields in both positional and orientational information. The two methods are most similar for small functional groups, while for larger functional groups MCSS yields results consistent with GRID but superior in detail. These results are in accord with the somewhat different purposes for which the two methods were developed. GRID has been used mainly to introduce functionalities at specific positions in lead compounds, in which case the orientation is predetermined by the structure of the latter. The orientational information provided by MCSS is important for its use in the de novo design of large, multi-functional ligands, as well as for improving lead compounds.

Algorithms↗

PubMed2000

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose.

BACKGROUND: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). RESULTS: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. CONCLUSIONS: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.

Amino Acid Sequence↗

PubMed2000

Computational design of D-peptide inhibitors of hepatitis delta antigen dimerization.

Hepatitis delta virus (HDV) encodes a single polypeptide called hepatitis delta antigen (DAg). Dimerization of DAg is required for viral replication. The structure of the dimerization region, residues 12 to 60, consists of an anti-parallel coiled coil [Zuccola et al., Structure, 6(1998)821]. Multiple Copy Simultaneous Searches (MCSS) of the hydrophobic core region formed by the bend in the helix of one monomer of this structure were carried out for many diverse functional groups. Six critical interaction sites were identified. The Protein Data Bank was searched for backbone templates to use in the subsequent design process by matching to these sites. A 14 residue helix expected to bind to the D-isomer of the target structure was selected as the template. Over 200,000 mutant sequences of this peptide were generated based on the MCSS results. A secondary structure prediction algorithm was used to screen all sequences. and in general only those that were predicted to be highly helical were retained. Approximately 100 of these 14-mers were model built as D-peptides and docked with the L-isomer of the target monomer. Based on calculated interaction energies, predicted helicity, and intrahelical salt bridge patterns, a small number of peptides were selected as the most promising candidates. The ligand design approach presented here is the computational analogue of mirror image phage display. The results have been used to characterize the interactions responsible for formation of this model anti-parallel coiled coil and to suggest potential ligands to disrupt it.

Algorithms↗

PubMed1999

Computational approaches to structure-based ligand design.

The first computational structure-based drug design methods came into existence in the early 1980s and are, to an extent, still in their infancy. There have been a few successes to date. With dramatic increases in computer speed, improved accuracy in ligand scoring functions, and the advent of combinatorial chemistry, there promises to be many more. In addition, the virtual explosion in the amount of available sequence and structural information has increased the need to develop these computational techniques to exploit this vast body of information. In this review, recent advances in computational methods for database searching and docking, de novo drug design, and estimation of ligand binding affinities are discussed.

Combinatorial Chemistry Techniques↗

PubMed1997

Use of the multiple copy simultaneous search (MCSS) method to design a new class of picornavirus capsid binding drugs.

A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) method and a simple scheme for joining MCSS functional group sites was applied to the binding pocket of P3/Sabin poliovirus and rhinovirus 14. The MCSS method determines where specific functional (chemical) groups have local potential energy minima in the binding site. Before the virus application, test calculations were run to determine the optimal set of input parameters to be used in evaluating the MCSS results. The MCSS minima are analysed and selected minima are connected with (CH2)n linkers to form candidate ligands, whose structures are optimized in the binding site. Estimates of the binding strength were made for the ligands and compared with those for known drugs. The results indicate that the proposed ligands should bind to P3/Sabin poliovirus at least as well as the best of the existing drugs, and that they should also bind to P1/Mahoney poliovirus and rhinovirus 14. A detailed comparison of the poliovirus and rhinovirus binding pockets and an analysis of drug binding specificity is presented.

Algorithms↗

PubMed1996

Ionic interactions in crystalline bovine pancreatic ribonuclease A.

Isomorphous crystals (space group P3(2)21) of bovine pancreatic ribonuclease A (RNase A) were prepared at a pH of 5.5 in a series of high salt conditions, where both the nature of the ions and the ionic strength varied: 80% ammonium sulfate (mu = 12.5); 8 M sodium formate (mu = 8.0); 3 M NaCl, 30% ammonium sulfate (mu = 7.0); 3 M CsCl, 30% ammonium sulfate (mu = 7.0); and 2.5 M NaCl, 3.3 M sodium formate (mu = 5.8). These structures were independently refined to a resolution of 2.0 A or better with R-factors that range from 16.1% to 17.5%. A comparison of these six structures and the monoclinic crystal form of RNase A grown from alcohol shows that changes in ionic strength do not alter the secondary or tertiary structure and that there are no significant changes in intramolecular salt bridges. These findings support the notion that structures determined from crystals grown in high salt are representative of the overall structural and electrostatic features present under physiological conditions. While little effect was observed on the main chain conformation, several residues adopted different side chain conformations and altered hydrogen-bonding patterns, either as result of direct anion binding or more subtle indirect effects. Changes in the ionic composition of the mother liquor allowed for the occupancy of the active site with different anions. The direct observation of active site-bound chloride and formate anions supports the proposal that these species act as true competitive inhibitors of RNase A and not through nonspecific electrostatic effects. The identification of bound formate anions allowed for an experimental validation of computational-based functional group mapping techniques and suggests a useful modification to these approaches. Electrostatic surface potential calculations identify a nearly continuous band of positive potential, consistent with an extended binding site for polynucleotide ligands and substrates. The majority of these residues are not involved in salt bridges, which may facilitate binding to extended polynucleotide substrates. Selection of the appropriate solvent conditions results in an unoccupied active site, which will allow this crystal form to be used for the crystallographic study of productive ligand-binding modes.

Amino Acid Sequence↗

PubMed1996

Comparison of experimental and computational functional group mapping of an RNase A structure: implications for computer-aided drug design.

One relatively new computational approach to the drug discovery process involves calculating functional group maps of a target structure. Experimental functional group mapping techniques have also recently emerged. In this paper, the structure of RNase A with two bound formates (i.e. carboxylate functionalities) is used as a model system to test the computational methodology. Functional group maps of the RNase A structure were calculated using the Multiple Copy Simultaneous Search (MCSS) method and compared with experimentally determined formate and water positions. The calculations indicate that the protonation state of active-site histidines determines the ability of the enzyme to bind formate. The results also suggest an ordered binding mechanism for the two formates. An improved strategy for using the MCSS method to design new candidate ligands is discussed.

Animals↗

PubMed1995

Use of a minimum perturbation approach to predict TIM mutant structures.

A minimum perturbation conformational search approach is used to model the structures of the yeast triosephosphate isomerase (TIM) single mutant in which the catalytic base Glu165 is changed to Asp, and the double mutant in which Glu165 is changed to Asp and Ser96 to Pro. In chicken TIM this double mutant is referred to as a pseudo-revertant because some of the catalytic activity lost due to the first mutation is regained when the second mutation occurs. Three minimum energy structures were calculated for the Asp165 conformation in the yeast TIM single mutant and another three for the double mutant. One of the calculated minimum energy conformations for Asp165 in the E165D structure agrees well with the X-ray structure. However, this conformation is not that of the lowest energy and is not one of the three most common conformers for Asp found by Ponder and Richards. This suggests that when an amino acid is introduced it may not be able to conform to the more general rules that apply to protein structures of evolutionary origin. While the van der Waals energy largely determines the allowed minima, the relative ranking of the final minima is determined by electrostatic effects and can therefore be affected by the inclusion of crystal waters in the calculation. When the E165D calculation is repeated with an active-site water molecule fixed in its E165D X-ray structure position, the relative ranking of the minima shifts and the X-ray conformation for Asp165 is the lowest interaction energy conformer. Two of the E165D calculated minimum energy structures are essentially identical to two of the S96P/E165D minima. All of the calculated minima for both the E165D and S96P/E165D mutants position the Asp side chain such that the anti-orbital, and not the more basic syn-orbital, of the carboxylate would be utilized for proton abstraction. This observation may explain why the chicken TIM S96P/E165D mutant, for which the X-ray structure indicates that the syn-orbital is used, is a pseudo-revertant while the yeast TIM double mutant is not; no X-ray structure is available for the latter. The multiplicity of minima found in the present analysis makes clear that predicting the exact orientation of a single side chain is not as simple as might be expected.

Amino Acid Sequence↗

PubMed1994

Crystal structure of the K12M/G15A triosephosphate isomerase double mutant and electrostatic analysis of the active site.

The crystal structure of the yeast triosephosphate isomerase (TIM) double mutant K12M/G15A has been solved to 2 A by X-ray diffraction, and the effects of changing the positively charged lysine to the neutral methionine have been analyzed. The mutant enzyme was crystallized in the presence of the tight-binding inhibitor phosphoglycolohydroxamate, under standard conditions for obtaining crystals of the enzyme-inhibitor complex. The crystals obtained were of the same crystal form as the unliganded wild-type enzyme. The three-dimensional structure confirms that the Lys-12 to Met mutation prevents the enzyme from binding substrate and reveals that the reason is electrostatic and not steric. The substrate-binding loop is in its open position and the Met side chain points away from the active site. Overall, the mutant structure is very similar to that of the wild-type unliganded enzyme. The electrostatic potential at the active site of the mutant enzyme is, however, very different from that of the wild type. It has been postulated previously that Lys-12 may play a role in stabilizing the negative charge in the transition state. This K12M/G15A structure suggests that the active-site Lys, which is strictly conserved, is required for TIM to be able to bind its dianionic substrate.

Amino Acid Sequence↗

PubMed1994

Crystal structure of the mutant yeast triosephosphate isomerase in which the catalytic base glutamic acid 165 is changed to aspartic acid.

The three-dimensional structure of the E165D mutant of the glycolytic enzyme yeast triosephosphate isomerase has been determined by X-ray diffraction at a nominal resolution of 2 A. For crystallization, the mutant enzyme was complexed with the tight-binding intermediate analog, phosphoglycolohydroxamate. Comparison with the structure of the wild-type enzyme reveals that, as originally intended, replacement of the catalytic base Glu-165 with the shorter side chain of aspartic acid has increased the distance between the base and the intermediate analog by 1 A. In addition, the catalytic base is oriented in the E165D structure so as to use the anti orbital of the carboxylate for proton abstraction; in the structure of the wild-type enzyme, the syn orbital is oriented for this purpose. It has been hypothesized that the 1000-fold loss in catalytic activity for this mutant triosephosphate isomerase is due either to the use of the less basic anti orbital for proton transfer or to the greater distance between the base and the substrate. The structure of yeast E165D triosephosphate isomerase suggests that both distance and orientation factors contribute to the loss of activity in the mutant enzyme and, therefore, that both factors contribute to the catalytic efficiency of the wild-type enzyme.

Amino Acid Sequence↗