Michael Cory

The discovery of potent cRaf1 kinase inhibitors.

A series of benzylidene-1H-indol-2-one (oxindole) derivatives was synthesized and evaluated as cRaf-1 kinase inhibitors. The key features of the molecules were the donor/acceptor motif common to kinase inhibitors and a critical acidic phenol flanked by two substitutions. Diverse 5-position substitutions provided compounds with low nanomolar kinase enzyme inhibition and inhibited the intracellular MAPK pathway.

Molecular diversity in chemical databases: comparison of medicinal chemistry knowledge bases and databases of commercially available compounds.

A molecular descriptor space has been developed which describes structural diversity. Large databases of molecules have been mapped into it and compared. This analysis used five chemical databases, CMC and MDDR, which represent knowledge bases containing active medicinal agents, ACD and SPECS, two databases of commercially available compounds, and finally the Wellcome Registry. Together these databases contained more than 300,000 structures. Topological indices and the free energy of solvation were computed for each compound in the databases. Factor analysis was used to reduce the dimensionality of the descriptor space. Low density observations were deleted as a way of removing outliers, which allowed a further reduction in the descriptor space of interest. The five databases could then be compared on an efficient basis using a metric developed for this purpose. A Riemann gridding scheme was used to subdivide the factor space into subhypercubes to obtain accurate comparisons. Most of the 300,000 structures were highly clustered, but unique structures were found. An analysis of overlap between the biological and commercial databases was carried out. The metric provides a useful algorithm for choosing screening sets of diverse compounds from large databases.

TOWARD ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY WITH THE T268G MUTANT OF HUMAN CARBOXYPEPTIDASE A1 AND NOVEL IN VIVO STABLE PRODRUGS OF METHOTREXATE

Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stablein vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-α-phenylalanine). Thus, thek cat/K m value for the wild type hCPA1 with MTX-α-phenylalanine is 0.44 μm −1 s−1, andk cat/K m values for hCPA1-T268G with MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine are 1.8 and 0.16 μm −1 s−1, respectively. The cytotoxic efficiency of hCPA1–268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with thein vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.

DOCKing ligands into receptors: The test case of α-chymotrypsin

Abstract Structures of 103 ligands previously tested as inhibitors of chymotrypsin catalysis were docked into the active site of the enzyme by use of the dock computer program. The goodness of fit was evaluated according to an approximate Lennard-Jones potential scoring routine. A statistical analysis indicated that dock correctly ranked the database when viewed in terms of a contingency table of four categories: true positives, false positives, true negatives, and false negatives. Eight of the top ten scoring molecules in the computerized docking procedure had been previously reported to be effective competitive inhibitors of chymotrypsin. This agreement between the computer predictions and experimental observations was encouraging and suggests that the dock computer program may be useful in evaluating other receptors for potential binding ligands.

MATCHMOL, an interactive computer graphics procedure for superposition of molecular models

Abstract A procedure that facilitates the interactive superposition of computer models of molecules was written. This procedure, a rigid body fit, used a fitting algorithm described by McLachlan and the molecular graphics facilities provided by the NIH - PROPHET system. The flexibility of the PROPHET molecule data type allows easy generation of hypothetical atoms (pseudo-atoms) which can serve as positional descriptors of putative pharmacophores. Definition of pseudoatoms which represent the centre of polycyclic aromatic chromophores and the superposition of two DNA binding compounds is used as an example of an application of the MATCHMOL procedure.

Chapter 28. Quantitative Structure Activity Relationships Applied to Drug Design

Publisher Summary This chapter discusses the research on the application of quantitative structure–activity relationship (QSAR) studies to drug design. QSAR studies of drug metabolism and distribution have been discussed in this chapter. The chapter mentions various methods by which the three dimensional conformation of the molecule is used to generate descriptors. These techniques include conformational energy minimization and exhaustive computerized search of conformational space with computation of ligand and receptor geometry. Mathematical methods researched on series design, including criteria for the measurement of the suitability of a proposed series and two-dimensional mapping of descriptors. The pattern recognition technique of adaptive least squares is applied to the discrimination of categorical biological data. Various parameters are argued upon. Partition coefficient is expressed either as log P or is still the most important parameter considered in developing QSAR relationships. It is rather tedious to measure log P by the solvent partition method, so recent workers have concentrated upon the correlation between partition coefficients and other accessible physical parameters. Reverse phase HPLC has been correlated with log P, using phenols, and with π, using nonionic pesticides. Structural diagrams are explained in the chapter. Applications of the QSAR techniques have been used to study inhibitors of dihydrofolate reductase (DHFR). Molecular properties, obtained from CND0/2 molecular orbital calculations, have been used to correlate the inhibition of DHFR by substituted quinazolines. Regression analysis was applied to quinazolines as inhibitors of DHFR from human and mouse leukemia cells. The antifibrinolytic activity of a series of arylacetic acids is correlated with lipophilicity. Estimation of lipophilicity by reversed phase thin layer chromatographic correlated poorly with the anti-fibrinolytic activity but tabulated π values correlate well. DNA-dependent inhibition of DNA polymerase, DNA binding, mammalian toxicity, and tumor selectivity of a series of bisguanylhydrazones is described in the chapter. The mammalian toxicity of these compounds is effectively correlated with chromatographically determined R m values. Other parameters are also considered.