Highly Multiplexed Kinase Profiling in Spleen with Targeted Mass Spectrometry Reveals Kinome Plasticity across Species.

Early attrition of drug candidates, including kinase inhibitors, often occurs due to issues that arise during preclinical safety and efficacy evaluation. This problem may be exacerbated by the fact that these studies might fail to consider the basic physiological differences that could exist between human patients and animal models. We report the development of a targeted mass spectrometry-based assay capable of monitoring >50 different kinases using peptides conserved in humans and the key preclinical species used in drug development (mouse, rat, dog, and cynomolgus monkey). These methods were then used to profile interspecies kinome variability in spleen with three of the current techniques used in targeted proteomics (MRM, PRM, and IS-PRM). IS-PRM provides the highest number of kinase identifications, and the results indicate that while this initial set of kinases exhibits high correlation between species for this tissue type, distinct species-specific differences do exist, especially within the cyclin-dependent kinase family. An initial screen in two species with the kinase inhibitor dasatinib in competition with the chemoproteomic kinase-binding probe XO44 demonstrated how the targeted methods can be further applied to study species-specific inhibitor occupancy profiles. Understanding such differences could help rationalize the findings of preclinical studies and have major implications for the selection of these animals as models in kinase drug development.

In Silico Design and in Vitro Characterization of Universal Tyrosine Kinase Peptide Substrates.

A majority of the 90 human protein tyrosine kinases (PTKs) are understudied "orphan" enzymes with few or no known substrates. Designing experiments aimed at assaying the catalytic activity of these PTKs has been a long-running problem. In the past, researchers have used polypeptides with a randomized 4:1 molar ratio of glutamic acid to tyrosine as general PTK substrates. However, these substrates are inefficient and perform poorly for many applications. In this work, we apply the KINATEST-ID pipeline for artificial kinase substrate discovery to design a set of candidate "universal" PTK peptide substrate sequences. We identified two unique peptide sequences from this set that had robust activity with a panel of 15 PTKs tested in an initial screen. Kinetic characterization with seven receptor and nonreceptor PTKs confirmed these peptides to be efficient and general PTK substrates. The broad scope of these artificial substrates demonstrates that they should be useful as tools for probing understudied PTK activity.

A Fluorescence Polarization Biophysical Assay for the Naegleria DNA Hydroxylase Tet1.

The discovery of the 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (Tet) protein family was an important advancement in our understanding of DNA-modified epigenetics. Potent inhibitors of these proteins are greatly desired for both the understanding of the functions of these enzymes and to serve as eventual therapeutic leads. So far, the discovery of such small molecules with high affinity has been quite limited. Original tools to screen for activity are greatly needed in order to accelerate this process. Here we present a novel fluorescent probe, and the results of a fluorescence polarization-based binding assay for Naegleria Tet1, a homologue to mammalian Tet. A fluorescence polarization-based competition assay was also established and applied to the rapid and quantitative measurement of the binding affinity of the cofactor αKG and several known Tet1 inhibitors.

Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening.

Jumonji C domain-containing histone demethylases (JHDMs) are epigenetic proteins capable of demethylating methylated lysine residues on histones proteins and for which high-quality chemical probes and eventual therapeutic leads are highly desirable. To expand the extent of known scaffolds targeting JHDMs, we initiated an unbiased high-throughput screening approach using a fluorescence polarization (FP)-based competitive binding assay we recently reported for JHDM1A (aka KDM2A). In total, 14,400 compounds in the HitFinder collection v.11 were screened, which represent all the distinct skeletons of the Maybridge Library. An eventual three compounds with two new scaffolds were discovered and further validated, which not only show in vitro binding for two different JHDMs, JHDM1A and JMJD2A (aka KDM4A), but also induce hypermethylation of their substrate in cells. These represent novel scaffolds as JHDM inhibitors and provide a basis for future optimization of affinity and selectivity.

Development of substrate-selective probes for affinity pulldown of histone demethylases.

JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of "methylation state" and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the "methylation state" and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators.

Discovery and initial structure-activity relationships of N-benzyl tricyclic indolines as antibacterials for methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent drug resistant bacteria. In 2012, over 11,000 fatalities in the United States were directly attributable to MRSA. In an effort to develop novel structural and mechanistic classes of antibacterial agents to fight against MRSA, we have optimized a hit compound, Of4, previously discovered in a screening campaign of a bio-inspired polycyclic indoline library previously developed in our lab. We took advantage of our concise and versatile synthetic strategy to conduct initial structure-activity relationship studies of Of4, and we now report the discovery of compound 4k as a more potent antibacterial agent against S. aureus. Compound 4k also displayed equivalent activity in four MRSA and a methicillin-susceptible strains while demonstrating an improved mammalian cytotoxicity profile compared to Of4. Interestingly, 4k shares the same tricyclic indoline core as Of1, a ß-lactam-selective resistance-modifying agent, but harbors a distinct modification pattern conferring unique bioactivity. This phenomenon is reminiscent of many bioactive natural products.