HTS in the new millennium: the role of pharmacology and flexibility.

Over the past decade, high throughput screening (HTS) has become the focal point for discovery programs within the pharmaceutical industry. The role of this discipline has been and remains the rapid and efficient identification of lead chemical matter within chemical libraries for therapeutics development. Recent advances in molecular and computational biology, i.e., genomic sequencing and bioinformatics, have resulted in the announcement of publication of the first draft of the human genome. While much work remains before a complete and accurate genomic map will be available, there can be no doubt that the number of potential therapeutic intervention points will increase dramatically, thereby increasing the workload of early discovery groups. One current drug discovery paradigm integrates genomics, protein biosciences and HTS in establishing what the authors refer to as the "gene-to-screen" process. Adoption of the "gene-to-screen" paradigm results in a dramatic increase in the efficiency of the process of converting a novel gene coding for a putative enzymatic or receptor function into a robust and pharmacologically relevant high throughput screen. This article details aspects of the identification of lead chemical matter from HTS. Topics discussed include portfolio composition (molecular targets amenable to small molecule drug discovery), screening file content, assay formats and plating densities, and the impact of instrumentation on the ability of HTS to identify lead chemical matter.

Yeast protein geranylgeranyltransferase type-I: steady-state kinetics and substrate binding.

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in Leu or Phe by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). A kinetic study of the alkylation reaction was conducted with a continuous assay based on the fluorescence enhancement that accompanies geranylgeranylation of dansyl-GCIIL. The kinetic constants k(cat) = 0.34 +/- 0.01 s(-1), K(M)(G) = 0.86 +/- 0.05 microM for GGPP, and K(M)(D) = 1.6 +/- 0.1 microM for dansyl-GCIIL were calculated from initial rates measured at varying concentrations of the substrates. Inhibitor studies were conducted with dead-end inhibitors for GGPP and the peptide substrate. Double reciprocal plots for the peptide mimic Cys-AMBA-Leu gave a competitive pattern when plotted against varying concentrations of dansyl-GCIIL and an uncompetitive pattern against GGPP. Similar plots for 1-phosphono-(E,E,E)-geranylgeraniol, a dead-end inhibitor for GGPP, gave a competitive double reciprocal plot for varied concentrations of GGPP and induced potent substrate inhibition by dansyl-GCIIL when dansyl-GCIIL was the varied substrate. The dissociation constant (K(D)) for the PGGTase-I x GGPP complex was 120 +/- 20 nM. These results are consistent with an ordered binding mechanism for PGGTase-I where GGPP adds before peptide.

Phosphonate and alpha-fluorophosphonate analogue probes of the ionization state of pyridoxal 5'-phosphate (PLP) in glycogen phosphorylase.

To investigate the role of the essential cofactor pyridoxal phosphate in rabbit muscle glycogen phosphorylase catalysis, two phosphonate analogues of pyridoxal phosphate, 5'-deoxypyridoxal 5'-methylenephosphonic acid and 5'-deoxypyridoxal 5'-difluoromethylenephosphonic acid, have been prepared and reconstituted into apophosphorylase b. UV/Vis spectroscopic and 31P and 19F NMR studies confirmed the successful reconstitution and revealed significant changes in phosphate environment upon nucleotide activation. Both such reconstituted enzymes had activities of approximately 25%-30% of that observed in the native enzyme, while K(m) values were similar to those of the native enzyme. Very similar dependences upon pH of Vmax, K(m), and Vmax/K(m) were found for the two reconstituted enzyme derivatives and the native enzyme despite the considerable difference in phosphonic acid pKa values. These results suggest that pyridoxal phosphate does not function as an essential acid/base catalyst in glycogen phosphorylase; rather they suggest that the cofactor phosphate moiety remain dianionic throughout catalysis and functions as an essential dianion. Mechanistic implications of these findings are discussed.

Yeast protein geranylgeranyltransferase type-I: overproduction, purification, and characterization.

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in leucine by the C20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). The Saccharomyces cerevisiae genes encoding the alpha (RAM2) and beta (CDC43) subunits of PGGTase-I were translationally coupled by overlapping the RAM2-CDC43 stop-start codons and by locating a ribosome-binding site near the 3' end of RAM2. Recombinant PGGTase-I was overproduced in Escherichia coli to give approximately 8% of total cellular protein and purified 12-fold to > 95% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The purified heterodimer contained alpha- and beta-subunits with molecular masses of 34 and 42 kDa, respectively. A continuous fluorescence assay was developed to measure PGGTase-I activity. The recombinant enzyme showed maximal activity at pH 7.5 and required both Mg2+ and Zn2+. Michaelis constants for GGPP (1.0 microM) and dansyl-Gly-Cys-Ile-Ile-Leu (2.4 microM) were similar to those reported for yeast protein farnesyltransferase (PFTase) with farnesyl diphosphate and dansyl-Gly-Cys-Val-Ile-Ala; Vmax = 0.20 mumol min-1 mg-1 for recombinant yeast PGGTase-I was similar to that reported for yeast PFTase.