Heidi E. Brown

Amides of xanthurenic acid as zinc-dependent inhibitors of Lp-PLA(2).

AX10185, the phenyl amide of xanthurenic acid, was found to be a sub-100nM inhibitor of Lp-PLA(2). However, in the presence of EDTA the inhibitory activity of AX10185 was extinguished while the enzymatic activity of Lp-PLA(2) did not change. Subsequent metal screening experiments determined the inhibition to be Zn(2+) dependent. Structure-activity relationship studies indicated the presence of the 4-hydroxy group to be critical and selected substituted phenyl, polycyclic, and cycloaliphatic amides of xanthurenic acid to be well tolerated.

Amides of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid as zinc-dependent inhibitors of Lp-PLA2

AX10479, the phenyl amide of 4-hydroxy-8-methanesulfonylamino-quinoline-2-carboxylic acid, was identified as a Zn(2+)-dependent, 27nM inhibitor of human plasma Lp-PLA(2). Structure-activity relationship studies focused on the AX10479 2-phenylamide group identified equipotent cycloaliphatic amides, an enantioselective preference for chiral amides, and phenyl substitution patterns (e.g., 2-methyl-3-fluoro) that increased potency.

Monitoring native p38α:MK2/3 complexes via trans delivery of an ATP acyl phosphate probe.

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways.

Chemoproteomic Evaluation of Target Engagement by the Cyclin-Dependent Kinase 4 and 6 Inhibitor Palbociclib Correlates with Cancer Cell Response

Palbociclib is a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor approved for breast cancer that is estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative. We profiled palbociclib in cells either sensitive or resistant to the drug using an ATP/ADP probe-based chemoproteomics platform. Palbociclib only engaged CDK4 or CDK6 in sensitive cells. In resistant cells, no inhibition of CDK4 or CDK6 was observed, although the off-target profiles were similar in both cell types. Prolonged incubation of sensitive cells with the compound (24 h) resulted in the downregulation of additional kinases, including kinases critical for cell cycle progression. This downregulation is consistent with cell cycle arrest caused by palbociclib treatment. Both the direct and indirect targets were also observed in a human tumor xenograft study using the COLO-205 cell line in which phosphorylation of the retinoblastoma protein was tracked as the pharmacodyanamic marker. Together, these results suggest that this probe-based approach could be an important strategy toward predicting patient responsiveness to palbociclib.

Synthesis and structure-activity relationship of (1-halo-2-naphthyl) carbamate-based inhibitors of KIAA1363 (NCEH1/AADACL1).

KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC(50) value of 1.2 μM and was shown using ESI-MS to carbamylate the catalytic residue Ser(191). SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice.

Profiling Native Kinases by Immuno-Assisted Activity-Based Profiling

The protocols in this unit describe efficient and cost‐effective approaches to determine the interaction of small‐molecule inhibitors with native kinases, and also analyze the interactions between kinases and their binding partners in a cellular setting. The combined attributes of activity‐based probes and western blotting procedures provide for quantitative measurement of inhibitor efficacy, isoform selectivity, and post‐translational modifications. We further demonstrate the ability to identify protein‐protein interactions between a probe‐labeled protein and its noncovalent binding partners. Curr. Protoc. Chem. Biol. 5:213‐226

Correction: Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

[This corrects the article DOI: 10.1371/journal.pone.0152934.].

Abstract B23: Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors

Kinases are an important class of targets for a number of therapeutic indications, and currently, kinase inhibitors constitute a significant fraction of recently approved drugs, particularly for cancer. Using a probe-based chemoproteomics platform, we have demonstrated the utility of the platform to monitor the interactions between inhibitors and native kinases. Herein, we profile kinase inhibitors in cell lines either sensitive or resistant to the compound for various periods of time. We observe that at early time points, inhibitor profiles for a compound are qualitatively similar in both the sensitive and resistant cells, suggesting the compound is capable of engaging its direct targets in both cell types. In contrast, at later time points, additional kinases are observed affected by compound treatment. We infer that these kinases do not directly bind the compound but rather reflect secondary effects that result from the engagement of the primary targets (pathway effects). Significantly, the pathway effects are only observed in the sensitive cells and not the resistant cells. Kinases affected through these pathway effects include kinases known to be critical for cell cycle progression including CDK1, Aurora A and B, PLK1, and MASTL. Finally, we demonstrate that during the treatment of tumor-bearing mice with an inhibitor, in addition to inhibition of the direct targets, pathway effects are observed recapitulating the results from the cell-based studies. Citation Format: Tyzoon K. Nomanbhoy, Heidi E. Brown, Jiangyue Wu, Subha Vogeti, Arwin Aban, Wendy Grant, Alemayehu Senait, Shuzhen Wu, Christa Dias, Geeta Sharma. Chemoproteomic profiling of native kinases during the treatment of cells with kinase inhibitors. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B23.