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Biomedical subjects

M C Nicklaus

Publications and source records attributed to M C Nicklaus.

At least 19 recordsLinked to original sources
PubMed2000

Interactions of conformationally biased north and south 2'-fluoro-2', 3'-dideoxynucleoside 5'-triphosphates with the active site of HIV-1 reverse transcriptase.

Molecular dynamics simulations of a ternary complex of HIV-1 reverse transcriptase (RT), double-stranded DNA, and bound dideoxynucleoside-5'-triphosphate (RT-DNA-ddNTP), utilizing the ddNTPs ddATP, betaFddATP, and alphaFddATP, explain the experimentally observed order of potency of these 5'-triphosphates as inhibitors of RT: ddATP > betaFddATP > alphaFddATP. On the basis of RT's known preference to bind the incoming dNTP (or ddNTP) with a north conformation at the polymerase site, alphaFddATP, which in solution prefers almost exclusively a north conformation, was predicted to be the most potent inhibitor. However, Tyr115, which appears to function as a steric gate to preclude the binding of ribonucleoside 5'-triphosphates, prevents the effective binding of alphaFddATP in its preferred north conformation. The south-biased betaFddATP, while able to bind to RT without hindrance by Tyr115, has to pay a high energy penalty to be flipped to the active north conformation at the polymerase site. Finally, the more flexible and less conformationally biased ddATP is able to switch to a north conformation at the RT site with a smaller energy penalty than betaFddATP. These results highlight the opposite conformational preferences of HIV-1 RT for alphaFddATP and betaFddATP and help establish conformational guidelines for optimal binding at the polymerase site of this enzyme.

Anti-HIV Agents↗
PubMed2000

Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies.

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.

Adenosine↗
PubMed2000

Identification of HIV-1 integrase inhibitors via three-dimensional database searching using ASV and HIV-1 integrases as targets.

Integration of viral DNA into the host cell genome is a critical step in the life cycle of HIV. This essential reaction is catalyzed by integrase (IN) through two steps, 3'-processing and DNA strand transfer. Integrase is an attractive target for drug design because there is no known cellular analogue and integration is essential for successful replication of HIV. A computational three-dimensional (3-D) database search was used to identify novel HIV-1 integrase inhibitors. Starting from the previously identified Y3 (4-acetylamino-5-hydroxynaphthalene-2,7-disulfonic acid) binding site on the avian sarcoma virus integrase (ASV IN), a preliminary search of all compounds in the nonproprietary, open part of the National Cancer Institute 3-D database yielded a collection of 3100 compounds. A more rigorous scoring method was used to rescreen the 3100 compounds against both ASV IN and HIV-1 IN. Twenty-two of those compounds were selected for inhibition assays against HIV-1 IN. Thirteen of the 22 showed inhibitory activity against HIV-1 IN at concentrations less than 200 microM and three of them showed antiviral activities in HIV-1 infected CEM cells with effective concentrations (EC50) ranging from 0.8 to 200 microM. Analysis of the computer-generated binding modes of the active compounds to HIV-1 IN showed that simultaneous interaction with the Y3 site and the catalytic site is possible. In addition, interactions between the active compounds and the flexible loop involved in the binding of DNA by IN are indicated to occur. The structural details and the unique binding motif between the HIV-1 IN and its inhibitors identified in the present work may contribute to the future development of IN inhibitors.

Avian Sarcoma Viruses↗
PubMed1999

Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions.

A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position -2 and G at position +6 were observed for the top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2. The VP-16-induced top2(S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-1, whereas the most reversible sites showed no base preference at positions -1 or -2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser740 --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.

Amino Acid Substitution↗
PubMed1998

Pharmacophores in drug design and discovery.

Compounds containing a specific pharmacophore--the minimum structural features necessary for enzyme binding--can be retrieved from a database such as the National Cancer Institute repository by means of three-dimensional (3D) searching, which allows the retrieval of all compounds possessing a specified set of atoms with a given 3D geometry. The ways in which pharmacophores can be found and characterized and the details of the 3D searching methods are described. Elaboration of compounds found in such searches and their subsequent development as lead drugs is also discussed.

Animals↗
PubMed1997

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.

Caffeic Acids↗
PubMed1997

Discovery of HIV-1 integrase inhibitors by pharmacophore searching.

Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152, compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206,876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 microM and are promising lead compounds for further HIV-1 integrase inhibitor development.

4-Hydroxycoumarins↗
PubMed1997

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

Anti-HIV Agents↗
PubMed1996

Discovery of novel, non-peptide HIV-1 protease inhibitors by pharmacophore searching.

Fifteen novel non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of the NCI DIS 3D database. The pharmacophore query used in the search was derived directly from the X-ray determined structures of protease/inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, NSC 32180, a "dimer" of 4-hydroxycoumarin, and NSC 117027, a "tetramer" of 2-hydroxy quinone, had ID50 values of 0.32 and 0.75 microM for HIV-1 protease inhibition, respectively, and two other inhibitors had ID50 values close to 1 microM. Among the potent inhibitors, NSC 158393 not only demonstrated activity against HIV-1 protease (ID50 1.7 microM) but also exhibited promising antiviral activity in HIV-1-infected CEM-SS cells (EC50 = 11.5 microM). Validation of the pharmacophore used in the search was accomplished by conformational analysis. The binding modes of the most potent inhibitor found in our studies, NSC 32180, were predicted employing docking and molecular dynamics techniques.

Amino Acid Sequence↗
PubMed1995

Conformational changes of small molecules binding to proteins.

Flexible molecules change their conformation upon binding to a protein. This was shown by the analysis of small molecules whose structures have been determined by X-ray crystallography of both the pure compound and the compound bound to a protein. Thirty-three compounds present both in the Cambridge Structural Database and the Brookhaven Protein Data Bank were analyzed, and both were compared with the global energy minimum conformation calculated by the molecular mechanics program CHARMm. It was found that the conformation bound to the protein differs from that in the crystal structure and also from that of the global energy minimum, and the degree of deformation depends upon the number of freely rotatable bonds in the molecule. Analysis of the conformational energies of the flexible molecules showed that, for most of those compounds, both the crystal and the protein-bound conformations are energetically well above the global minimum, and, in many cases, not even in any local energy minimum. Semi-empirical calculations performed for a select number of structures, using both the AM1 and PM3 hamiltonians, confirmed these results. These findings are discussed as to their impact upon contemporary methods of drug design.

Crystallography↗
PubMed1994

Conformationally locked nucleoside analogues. Synthesis of dideoxycarbocyclic nucleoside analogues structurally related to neplanocin C.

The glycon moiety of nucleosides in solution is known to exist in a rapid dynamic equilibrium between extreme northern and southern conformations as defined by the pseudorotation cycle. The concept of preparing rigid nucleoside analogues with the glycon conformation locked in one of these two extremes was tested with the synthesis of some cyclopropane-fused dideoxycarbocyclic nucleosides, similar to the well-known class of anti-HIV active dideoxynucleosides. The new compounds described here are dideoxynucleoside analogues of the fermentation product neplanocin C (6) which exhibits a typical northern geometry for its 6-oxabicyclo[3.1.0]hexane pseudosugar moiety. However, in view of the lability of the epoxide ring in this system, the equivalent cyclopropane-fused bicyclo[3.1.0]hexane system was used instead to prepare the corresponding dideoxynucleoside analogues bearing all the common bases [(+/-)-9-13]. Due to the well-documented preference of unrestricted bicyclo[3.1.0]hexane systems to exist exclusively in a boat conformation, the resulting nucleosides are structurally locked in a typical northern conformation similar to that of neplanocin C. The locked northern conformation in these nucleosides remained unchanged in solution in the 20-80 degrees C temperature range according to variable temperature 1H NMR studies. For the synthesis of these compounds, racemic trans-1-[(benzyloxy)methyl]-4-hydroxybicyclo[3.1.0]hexane [(+/-)-18] was prepared by a samarium-promoted cyclopropanation reaction with the antecedent cyclopentenol. All of the bases were incorporated under Mitsunobu conditions and converted to the desired final products following a standard methodology. Anti-HIV evaluation revealed that only the adenosine analogue (+/-)-9 possessed enough activity to warrant resolution into its optical antipodes. This was realized by chiral HPLC chromatography to give the individual enantiomers (-)-32 and (+)-33. Adenosine deaminase was used to identify isomer (+)-33 as the enantiomer with the "natural" configuration which was solely responsible for the observed biological activity and toxicity of (+/-)-9. It is possible that the exclusive northern conformation adopted by these nucleosides reduces their substrate affinity for the various activating kinases, except in the case of the adenosine analogue.

Adenine↗
PubMed1994

Protein kinase C. Modeling of the binding site and prediction of binding constants.

A detailed examination of the mode of binding of phorbol esters to protein kinase C led to a model of the phorbol binding site in the enzyme. The efficacy with which various synthetic diacylglycerol analogs and ribonolactones are able to bind to this site was determined by means of semiempirical quantum mechanical calculations using PM3, and an estimate of the binding energy was made in each case. Sixteen synthetic analogs of 1,2-diacylglycerol and two natural products were studied, and their calculated energies of binding to this model were correlated with the measured Ki values. The binding energies calculated for this receptor model, together with solubility and entropy considerations, allow prediction through regressive fit of free energies of binding which correlate very well with the measured binding constants.

Binding Sites↗
PubMed1992

QSAR of conformationally flexible molecules: comparative molecular field analysis of protein-tyrosine kinase inhibitors.

Comparative Molecular Field Analysis (CoMFA) has been applied to a study of quantitative structure-activity relationships (QSAR) of conformationally flexible molecules. The relationship between three-dimensional structure and activity of 20 styrene derivatives which inhibit protein-tyrosine kinase was determined. A technique was developed that allows accurate prediction of the inhibitory activity of these molecules and identification in each case of the active conformation. The problem of multiple energetically acceptable conformations was approached in an iterative procedure. Use was made of the varying degrees of symmetry among the molecules. First, CoMFA QSAR models were developed using only those compounds that possess a symmetrical substituent pattern on the phenyl ring. These CoMFA models were then used to select the active conformers of the less symmetrical compounds in the set. Allowing multiple conformers for each compound in the dataset yielded higher crossvalidated r2 values and better predictivity of the QSAR models. Different probe atoms (C+, O-, neutral C) were explored, the O- probe atom exhibiting the highest selectivity in the conformer selection process.

Drug Design↗
PubMedDate not supplied

Chem-X and CAMBRIDGE. Comparison of computer generated chemical structures with X-ray crystallographic data.

The structures of a number of molecules as determined by X-ray crystallography have been compared with the structures for the same molecules as calculated by the 3D structure generation program, Chem-X. In the group of molecules examined, ChemModel produced structures that were essentially identical to those based upon X-ray data in 57% of the cases. The corresponding figure for the widely used alternative model builder, CONCORD, was 38%. The superior performance of ChemModel was due entirely to that program's ability to generate multiple structures covering the entire conformational space.

Binding Sites↗
PubMedDate not supplied

National Cancer Institute Drug Information System 3D database.

A searcheable database of three-dimensional structures has been developed from the chemistry database of the NCI Drug Information System (DIS), a file of about 450,000 primarily organic compounds which have been tested by NCI for anticancer activity. The DIS database is very similar in size and content to the proprietary databases used in the pharmaceutical industry; its development began in the 1950s; and this history led to a number of problems in the generation of 3D structures.

Antineoplastic Agents↗
PubMedDate not supplied

Molecular modeling in the discovery of drug leads.

The National Cancer Institute of the U.S.A. maintains a repository of about 500,000 chemicals which it has tested at some time for anticancer activity. As new chemotherapeutic targets present themselves, methods have been developed by which this large database can be re-examined without resorting to expensive high-volume biological screening. Electronic screening, the method described in this paper, begins with the identification of a target enzyme. The pharmacophore used by the enzyme in binding to substrates is identified, and then all compounds in the database that contain the pharmacophore are found. These compounds are then further filtered, for example, by physical properties such as solubility, and the relatively small number of compounds that survive are submitted for biological testing. This use of a primary electronic screen in the search for ligands of protein kinase C is described. The screen is very fast, and the method is quite generally applicable to different enzymes.

Antineoplastic Agents↗
PubMedDate not supplied

Computational chemistry on commodity-type computers.

A number of inexpensive computers were benchmarked with the ab initio program Gaussian 94, using both small standard test jobs and larger density functional (DFT) calculations. Several varieties of Pentium (x86) and Alpha CPU based systems were tested. Most of them were running under the open source code operating system Linux. They were compared with several workstations and supercomputers. The most powerful of today's commodity-type processors surpassed current supercomputers in speed. The choice of compilers and compilation options was often found to have a larger influence on job CPU times than details of the hardware. Especially on the x86 type machines, the jobs always ran faster the less memory (RAM) they were given. The fastest machine on a per-CPU basis was an Alpha/Linux system. For the DFT calculation, it was close to twice as fast as a Cray J90 supercomputer.

Chemistry↗