Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures.

Quantitative, Real-Time Measurements of Intracellular Target Engagement Using Energy Transfer.

Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement.

Emerging Approaches for the Identification of Protein Targets of Small Molecules - A Practitioners' Perspective.

Small-molecule (SM) leads in the early drug discovery pipeline are progressed primarily based on potency against the intended target(s) and selectivity against a very narrow slice of the proteome. So, why is there a tendency to wait until SMs are matured before probing for a deeper mechanistic understanding? For one, there is a concern about the interpretation of complex -omic data outputs and the resources needed to test these hypotheses. However, with recent advances in broad endpoint profiling assays that have companion reference databases and refined technology integration strategies, we argue that data complexity can translate into meaningful decision-making.

The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function.

The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity.

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2.

Quest for the Vulnerable Atheroma: Carotid Stenosis and Diametric Strain--A Feasibility Study.

The Bernoulli effect may result in eruption of a vulnerable carotid atheroma, causing a stroke. We measured electrocardiography (ECG)-registered QRS intra-stenotic blood velocity and atheroma strain dynamics in carotid artery walls using ultrasonic tissue Doppler methods, providing displacement and time resolutions of 0.1 μm and 3.

Development of a high-content screening assay panel to accelerate mechanism of action studies for oncology research.

Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth-inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays.

Development of an interactive Coronary Doppler Vibrometry system for detection of coronary artery disease.

Coronary artery disease is a deadly and costly condition and represents an important public health issue globally. Coronary Doppler vibrometry (CDV), a new noninvasive coronary artery stenosis detection technique, showed encouraging results in our recent clinical study. However, the CDV system required lengthy offline data analysis, thus it did not provide any feedback during examination on the quality of data, not to mention analysis results.

Transthoracic coronary Doppler vibrometry in the evaluation of normal volunteers and patients with coronary artery stenosis.

Coronary artery vibrometry is a new transthoracic Doppler ultrasound method for the detection of coronary artery stenosis. It detects audio-frequency vibrations generated by coronary artery luminal diameter reduction. We studied 31 patients with known or suspected stenosis using coronary artery vibrometry and quantitative coronary angiography and 83 normal volunteers.

Discovery and characterization of non-ATP site inhibitors of the mitogen activated protein (MAP) kinases.

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor.

Discovery of a novel small molecule binding site of human survivin.

Survivin is one of the most tumor-specific genes in the human genome and is an attractive target for cancer therapy. However, small-molecule ligands for survivin have not yet been described. Thus, an interrogation of survivin which could potentially both validate a small-molecule therapy approach, and determine the biochemical nature of any of survivin's functions has not been possible.

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.

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.

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.

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.

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.

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.

Blunt chest trauma victims: role of intravascular ultrasound and transesophageal echocardiography in cases of abnormal thoracic aortogram.

Purpose: The objective of our study was to use transesophageal echocardiography (TEE) and intravascular ultrasonography (IVUS) to evaluate their role in interrogating abnormal or equivocal findings seen on thoracic aortography performed on blunt chest trauma patients.

Methods: A prospective, nonblinded, observational, institutional review board-approved study of IVUS and TEE was conducted in acute blunt chest trauma victims with abnormal findings on thoracic aortograms. IVUS was performed with a 20-MHz catheter and TEE was performed with an omniplane probe.

Complex gel permeation assays for screening combinatorial libraries.

Gel permeation methods have been commonly used to screen combinatorial libraries synthesized on a solid support. We report here three screens of combinatorial libraries using gel permeation assays. These include a simple enzymatic assay to identify inhibitors of the influenza enzyme neuraminidase, and two more complex assays designed to screen for inhibitors of the interleukin-8 (IL-8)-IL-8 receptor and the urokinase-urokinase receptor interactions, respectively.

The incidence of pulmonary embolism in unexplained sudden cardiac arrest with pulseless electrical activity.

Purpose: The cause of many cases of sudden cardiac arrest from pulseless electrical activity is unknown. We hypothesized that pulmonary embolism was responsible for a substantial proportion of these cases and used transesophageal echocardiography to identify pulmonary embolism among patients with sudden cardiac arrest.

Subjects And Methods: We performed a prospective study at a tertiary care, university-operated county hospital, with a level 1 trauma center.