Biomedical subjects

Kenneth M Comess

Publications and source records attributed to Kenneth M Comess.

6 recordsLinked to original sources

PubMed2006

An ultraefficient affinity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF.

The authors describe the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosyn-thesis enzyme, MurF, by a novel affinity screening method. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively nonselective ligands, sometimes referred to as "nuisance" or "promiscuous" compounds. Ligands selective in their binding affinity for the MurF target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.

Anti-Bacterial Agents↗

PubMed2006

Kinase drug discovery by affinity selection/mass spectrometry (ASMS): application to DNA damage checkpoint kinase Chk1.

Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.

Amino Acid Sequence↗

PubMed2006

Development of sulfonamide compounds as potent methionine aminopeptidase type II inhibitors with antiproliferative properties.

We have screened molecules for inhibition of MetAP2 as a novel approach toward antiangiogenesis and anticancer therapy using affinity selection/mass spectrometry (ASMS) employing MetAP2 loaded with Mn(2+) as the active site metal. After a series of anthranilic acid sulfonamides with micromolar affinities was identified, chemistry efforts were initiated. The micromolar hits were quickly improved to potent nanomolar inhibitors by chemical modifications guided by insights from X-ray crystallography.

Aminopeptidases↗

PubMed2004

NMR-driven discovery of benzoylanthranilic acid inhibitors of far upstream element binding protein binding to the human oncogene c-myc promoter.

Reversal of aberrant gene expression that is induced by the proto-oncogene c-myc is likely to be effective for treating a variety of tumors that rely on this pathway for growth. One strategy to down-regulate the c-myc pathway is to target transcription factors that regulate its own expression. A host of proteins act in coordination to regulate c-myc expression and any one of them are theoretical targets for small-molecule therapy. Experimentally, it has been shown that the far upstream element (FUSE) binding protein (FBP) is essential for c-myc expression, and reductions in FBP levels both reduce c-myc expression and correlate with slower cell growth. FBP binds to ssDNA by capturing exposed DNA bases in a hydrophobic pocket. This suggests that a small molecule could be designed to occupy this pocket and inhibit FBP function. Using a variety of screening methodologies, we have identified ligands that bind to the DNA binding pockets of the KH domains of FBP. Gel shift analyses using full length FBP and a related transcription factor confirm that a small-molecule lead compound inhibits DNA binding in a specific manner. The benzoylanthranilic acid compounds described here represent leads in the design of FBP inhibitors that can serve as useful tools in the study of c-myc regulation and in the development of therapeutics that target the c-myc pathway.

Binding Sites↗

PubMed2004

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms.

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.

Apoptosis↗

PubMed2004

Affinity-based screening techniques for enhancing lead discovery.

Contemporary, rational small-molecule lead discovery methods, comprising target identification, assay development, high-throughput screening (HTS), hit characterization and medicinal chemistry optimization, dominate early-stage drug discovery strategies in many pharmaceutical companies. There is a growing disparity between the increasing cost of funding these methods and the decreasing number of new drugs reaching the market. New strategies must be adopted to reverse this trend. The use of genomics- and proteomics-based target discovery efforts can aid the process by dramatically increasing the number of novel, more highly validated targets entering the discovery process, but HTS must meet this increased demand with faster, cheaper technologies. Although activity-based screening strategies are typically efficient, allowing one scientist to interrogate tens of thousands of compounds per day, affinity-based screening strategies can allow much greater efficiency in the overall process. Affinity-based methods can play a role in both facilitating the screening of a greater number of targets and in efficiently characterizing the primary hits discovered.

Drug Design↗