Haiching Ma

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

Small-molecule protein kinase inhibitors are widely used to elucidate cellular signaling pathways and are promising therapeutic agents. Owing to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments that use these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we used functional assays to profile the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases. Quantitative analysis revealed complex and often unexpected interactions between protein kinases and kinase inhibitors, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can identify multitargeted inhibitors of specific, diverse kinases. The results have implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments involving kinase inhibitors.

The challenge of selecting protein kinase assays for lead discovery optimization.

Background: Protein kinases represent one of the most promising groups of drug targets owing to their involvement in such pathological conditions as cancer, inflammatory diseases, neural disorders, and metabolism problems. In the last few years, numerous pharmaceutical and biotech companies have established kinase high-throughput screening (HTS) programs, and the reagent and service industries for kinase assay platforms, kits, and profiling services have begun to thrive. Objective: The plethora of different assay formats available today poses a great challenge to scientists who want to select a technology that will be cost efficient, convenient to use, and have low false positive and false negative rates. Methods: In the current review, we summarize the most commonly used kinase assay methods in the drug discovery process, present the advantages and disadvantages of each of these methods, and discuss the challenges of discovering kinase inhibitors by using these technologies. Conclusions: The decision of selecting the assay formats for HTS or service platform for profiling should take into account not only the final goals of the screens but also the limitation of resources.

Nonclassical Nuclear Localization Signal Provides for High Efficiency, Nonviral Gene Transfer to Nondividing Endothelium

Nonclassical Nuclear Localization Signal Provides for High Efficiency, Nonviral Gene Transfer to Nondividing Endothelium

Chemical microarray: a new tool for drug screening and discovery

HTS with microtiter plates has been the major tool used in the pharmaceutical industry to explore chemical diversity space and to identify active compounds and pharmacophores for specific biological targets. However, HTS faces a daunting challenge regarding the fast-growing numbers of drug targets arising from genomic and proteomic research, and large chemical libraries generated from high-throughput synthesis. There is an urgent need to find new ways to profile the activity of large numbers of chemicals against hundreds of biological targets in a fast, low-cost fashion. Chemical microarray can rise to this challenge because it has the capability of identifying and evaluating small molecules as potential therapeutic reagents. During the past few years, chemical microarray technology, with different surface chemistries and activation strategies, has generated many successes in the evaluation of chemical–protein interactions, enzyme activity inhibition, target identification, signal pathway elucidation and cell-based functional analysis. The success of chemical microarray technology will provide unprecedented possibilities and capabilities for parallel functional analysis of tremendous amounts of chemical compounds.

Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells

We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5–1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10−4, these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.

Non-classical nuclear localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines

Gene transfer into CNS is critical for potential therapeutic applications as well as for the study of the genetic basis of neural development and nerve function. Unfortunately, lipid-based gene transfer to CNS cells is extremely inefficient since the nucleus of these post-mitotic cells presents a significant barrier to transfection. We report the development of a simple and highly efficient lipofection method for primary embryonic rat hippocampal neurons (up to 25% transfection) that exploits the M9 sequence of the non-classical nuclear localization signal of heterogeneous nuclear ribonucleoprotein A1 for targeting beta(2)-karyopherin (transportin-1). M9-assistant lipofection resulted in 20-100-fold enhancement of transfection over lipofection alone for embryonic-derived retinal ganglion cells, rat pheochromocytoma (PC12) cells, embryonic rat ventral mesencephalon neurons, as well as the clinically relevant human NT2 cells or retinoic acid-differentiated NT2 neurons. This technique can facilitate the implementation of promoter construct experiments in post-mitotic cells, stable transformant generation, and dominant-negative mutant expression techniques in CNS cells.

Nonviral gene therapy and its delivery systems.

Nonviral gene therapy has significant clinical potential, yet its therapeutic utility has been hindered by low transfection efficiency due to a combination of extracellular and intracellular barriers. Recent developments in formulation and delivery methodology have allowed a number of advances toward high efficiency gene delivery to various cell types and organs. In particular, the extracellular and intracellular pharmacokinetics of plasmid DNA trafficking are better understood in a number of cell systems. Using cationic lipid or polymers (often with receptor targeting), more than 10(5) plasmids can be delivered to a single cell. Endosomolytic agents promote endosome disruption, and include: weak bases, proton-sponge polymers, fusogenic peptides, viral particles, and photosensitizing compounds. Both classical and nonclassical nuclear localization signal (NLS) peptides have also been tested for enhancement of the probability of nuclear import events, a major rate-limiting step in DNA delivery to nondividing cells. For example, the M9 sequence from heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) protein, a nonclassical NLS, has been found to increase gene expression level by more than 10 to 150-fold in a variety of cell types. This review will concentrate on the current understandings of the basic mechanisms of nonviral gene delivery and new approaches in the field.

Conformational Analysis of the DFG-Out Kinase Motif and Biochemical Profiling of Structurally Validated Type II Inhibitors

Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as “classical DFG-out” conformations in order to distinguish them from conformations that have also been referred to as DFG-out in the literature but that do not have a fully formed allosteric pocket. We have completed a structural analysis of almost 200 small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that nonclassical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two data sets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity.

Assay Development for Histone Methyltransferases

Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-L-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpot(SM) platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC₅₀ values at a low μM range.

Adenovirus or HA-2 fusogenic peptide-assisted lipofection increases cytoplasmic levels of plasmid in nondividing endothelium with little enhancement of transgene expression.

Adenovirus‐assisted lipofection has been reported to increase transfection efficiency through mechanisms potentially involving endosome escape and/or nuclear targeting activity. Similarly, transfection with the viral fusogenic peptide HA‐2 of the influenza virus hemagglutinin can increase transfection efficiency. However, there are few studies examining the mechanism and intracellular trafficking of these viral and/or viral fusogenic peptide‐assisted lipofections.