Robin W. Spencer

High-throughput screening of historic collections: observations on file size, biological targets, and file diversity.

At Pfizer Central Research, high-throughput screening has been an important source of new leads for drug discovery for a decade. Our experience with over 150 high-throughput screens can address questions about necessary file size, how well particular biological targets fare (with particular reference to protein-protein interactions), and what file diversity means in practice.

Fluorescence-based continuous assay for the aspartyl protease of human immunodeficiency virus-1

5‐Dimethylaminonaphthalene‐1‐sulfonyl‐Ser‐Gln‐Asn‐Tyr‐Pro‐Ile‐Val‐Trp (Dns‐SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus‐1. In intact substrate, fluorescence of Trp (λex 290 nm, λem 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease‐catalyzed cleavage at the Tyr‐Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations <60 μM, initial reaction velocity increased as a linear function of substrate concentration, with k cat/K M= 9700 M−1s−1. Limited solubility and internal fluorescence quenching precluded a determination of K M for Dns‐SQNYPIVW, but this was clearly > 100 μM.

Inhibition of Helicobacter pylori Urease by Phenyl Phosphorodiamidates: Mechanism of Action

Helicobacter pylori urease is a nickel-containing enzyme that hydrolyzes urea to bicarbonate and ammonia. Andrews et al. (J. Am. Chem. Soc. 1986, 108, 7124) have shown that amides and esters of phosphoric acid are slow, tight-binding inhibitors of urease isolated from jack bean. We show that 4-substituted phenyl phosphorodiamidates (4-R-PhOP(=O)(NH2)2) are slow-binding inhibitors of H. pylori urease with no evidence of kinetic saturation. Their second-order rates of inhibition ki are strongly correlated with phenol pKa (e.g. R = NO2, ki = 2.5 x 10(5) M-1s-1; R = OMe, ki = 1.2 x 10(4) M-1s-1). The Bronsted beta for inhibition is 0.4, similar to that of model system SN2(P) reactions. Based on these observations, we suggest that urease inhibition is covalent but reversible, involving a common phosphoacyl enzyme intermediate.

Peptidyl O-acyl hydroxamates: Potent new inactivators of cathepsin B†

Peptidyl O-acyl hydroxamates having appropriate active-site recognition features are very potent time-dependent inhibitors of the cysteine proteinase cathepsin B. The inhibition is irreversible, and the inactivation rate is strongly dependent on peptide structure and correct positioning of the P1 amino acid carbonyl group. Lipophilic O-acyl groups provide the most rapid inactivators, as exemplified by the inhibitor O-mesitoyl N-benzyloxycarbonyl-L-phenylalanyl-L-alanine hydroxamate (kmax/Ki = 640,000 M-1s-1).

CP-70,030 and CP-75,998: The first non-peptide antagonists of bombesin and gastrin releasing peptide

Abstract CP-70,030 and CP-75,998 were identified in a screening program as compounds able to displace [125I]-gastrin releasing peptide (GRP) from its rat brain receptor. We describe here the syntheses of these compounds and their characterization as bonafide GRP antagonists.

NanoStore: A Concept for Logistical Improvements of Compound Handling in High-Throughput Screening

Small molecule screening, the systematic encounter of biology space with chemical space, has provoked the emergence of a whole industry that recreates itself by constant iterative improvements to this process. The authors describe an approach to tackle the problem for one of the most time-consuming steps in the execution of a screening campaign, namely, the reformatting of high-throughput screening test compounds from master plates to daughter assay plates used in the execution of the screen. Through an engineered storage procedure, they prepare plates ahead of the screening process with the respective compounds in a ready-to-use format. They show the biological inertness of the method and how it facilitates efficient recovery of compound activity. This uncoupling of normally interconnected processes provides time and compound savings, avoids repeated freeze-thaw cycles of compound solutions, and removes the problems associated with the DMSO sensitivity of certain assays types.

Physical parameters for brian uptake: optimizing log P, log D and pKa of T H A

Abstract Two positions on the acetylcholinesterase (AChE) inhibitor, 9-amino-1,2,3,4-tetrahydroacridine (THA), have been identified which can be modified to vary log P, log D and pKa values. Most importantly, these changes can be carried out without altering its AChE activity.

Inhibition of human leukocyte elastase (HLE) by novel bicyclic β-lactams

Abstract Novel 3,2,0 bicyclic β-lactam sulfenamides and selected carbon isosteres are inhibitors of human leukocyte elastase. The comparative biological activity and hydrolytic stability of these compounds are described.

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Abstract Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h. Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.

A General Strategy for the Synthesis of Eosin Fluorescein Energy Transfer Substrates for High Sensitivity Screening of Protease Inhibitors

Publisher Summary This chapter presents a general strategy for the synthesis of eosin fluorescein energy transfer substrates for high-sensitivity screening of protease inhibitors. In a study described in the chapter, peptide synthesis was done on 0.2 mmole scales, using 1.8 g of FMOC-Cys-Pepsyn KA resin, loaded as slurry. The column was placed onto the peptide synthesizer, and peptide synthesis was initiated using 90 min coupling cycles followed by 5 min capping cycles/0.5 M acetic anhydride. The amino acids were coupled directly as their pentafluorophenyl or, in the case of threonine, 3,4-dihydro-4-oxo-benzotriazine-3-oxy esters, using fourfold molar excess. The synthesis utilized the following side -chain protection: (1) Thr- and (2) His-. FMOC-gamma-amino butyric acid was combined with one equivalent each of 1-hydroxybenzotriazole and benzotriazol-l-yloxy tris phosphonium hexafluorophosphate as solids. Renin activity was measured by monitoring the change in fluorescence of a buffer containing 0.12 μg/ml renin, 100 mM NaCI, and 50 mM Na 2 HPO 4 /pH 6.8 with various amounts of fluorogenic substrate III. The buffer was excited at 485 nm, and the emission was read at 535 nm in an IDEXX FCA microtiter plate fluorimeter.