Quantifying biogenic bias in screening libraries.

In lead discovery, libraries of 10(6) molecules are screened for biological activity. Given the over 10(60) drug-like molecules thought possible, such screens might never succeed. The fact that they do, even occasionally, implies a biased selection of library molecules. We have developed a method to quantify the bias in screening libraries toward biogenic molecules. With this approach, we consider what is missing from screening libraries and how they can be optimized

The Chemical Basis of Pharmacology

ABSTRACT: Molecular biology now dominates pharmacology so thoroughly that it is difficult to recall that only a generation ago the field was very different. To understand drug action today, we characterize the targets through which they act and new drug leads are discovered on the basis of target structure and function. Until the mid-1980s the information often flowed in reverse: investigators began with organic molecules and sought targets, relating receptors not by sequence or structure but by their ligands. Recently, investigators have returned to this chemical view of biology, bringing to it systematic and quantitative methods of relating targets by their ligands. This has allowed the discovery of new targets for established drugs, suggested the bases for their side effects, and predicted the molecular targets underlying phenotypic screens. The bases for these new methods, some of their successes and liabilities, and new opportunities for their use are described. So dominant has the molecular biology view of pharmacology become that it is difficult to remember that even 25 years ago it was little more than an aspiration. Today we understand the activity of drugs and reagents first through the specific, clonable receptor molecules with which they interact. To understan

Structure-Based Optimization of a Non-\u3b2-lactam Lead Results in Inhibitors That Do Not Up-Regulate \u3b2-Lactamase Expression in Cell Culture

Bacterial expression of \u3b2-lactamases is the most widespread resistance mechanism to \u3b2 -lactam antibiotics. There is a pressing need for novel, non-\u3b2-lactam inhibitors of these enzymes. Our lead, compound 1, is chemically dissimilar to \u3b2 -lactams and is a noncovalent, competitive inhibitor of the enzyme. However, at 26 \u3bcM its activity is modest (Figure 1). Using the X-ray structure of the AmpC/1 complex as a template, 14 analogues were designed and synthesized. Among these, compound 10, had a Ki of 1 \u3bcM, 26-fold better than the lead. The structures of AmpC in complex with compound 10 and an analogue, compound 11, were determined by X-ray crystallography to 1.97 and 1.96 \uc5, respectively. Compound 10 was active in cell culture, reversing resistance to the third generation cephalosporin ceftazidime in bacterial pathogens expressing AmpC. In contrast to \u3b2-lactam-based inhibitors compound 10 did not up-regulate \u3b2-lactamase expression in cell culture but simply inhibited the enzyme expressed by the resistant bacteria. Its escape from this resistance mechanism derives from its dissimilarity to \u3b2 -lactam antibiotics

Structure-based discovery and in-parallel optimization of novelcompetitive inhibitors of thymidylate synthase

AbstractBackground:The substrate sites of enzymes are attractive targets for structurebased inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging.Results:In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and clocking studies led to a derivative of one of these, dansyltyrosine (Ki 65 μM. Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 μM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes.Conclusions:TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged

Energetic, structural, and antimicrobial analyses of β-lactam side chain recognition by β-lactamases

AbstractBackground: Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these β-lactams, most often through bacterial expression of β-lactamases, threatens public health. To understand how β-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because β-lactams form covalent adducts with β-lactamases. This has complicated functional analyses and inhibitor design.Results: To investigate the contribution to interaction energy of the key amide (R1) side chain of β-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine β-lactamases. Therefore, binding energies can be calculated directly from Ki values. The Ki values measured span four orders of magnitude against the Group I β-lactamase AmpC and three orders of magnitude against the Group II β-lactamase TEM-1. The acylglycineboronic acids have Ki values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of β-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of β-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to β-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 Å and 1.75 Å resolution; these structures suggest interactions that are important to the affinity of the inhibitors.Conclusions: Acylglycineboronic acids allow us to begin to dissect interaction energies between β-lactam side chains and β-lactamases. Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in β-lactam inhibitors or β-lactam substrates of serine β-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities. The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of anti-resistance compounds in this series

Colloidal Aggregation Causes Inhibition of G Protein-Coupled Receptors

Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field

Computational Biology and High Performance Computing 2000

Tutorial to be presented at Supercomputing 2000, Dallas TX, 6-10 November 2000.This work was supported by the Director, Office of Science, Office of Advanced Scientific computing Research, Mathematical, Information, and Computational Sciences Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF0009

Computational Biology and High Performance Computing 2000

Tutorial to be presented at Supercomputing 2000, Dallas TX, 6-10 November 2000.This work was supported by the Director, Office of Science, Office of Advanced Scientific computing Research, Mathematical, Information, and Computational Sciences Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF0009