ZIA BC 011471 (ZIA) | |||
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Title | Cell-free assay technologies for the identification of active compounds | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | O'Keefe, Barry | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $719,148 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Autoimmune Diseases (33.0%) Cancer (100.0%) Digestive Diseases (33.0%) |
Non Hodgkins Lymphoma (34.0%) | ||
Research Type | |||
Systemic Therapies - Discovery and Development Combinations of Localized and Systemic Therapies |
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Abstract | |||
This project resulted in the development high throughput screens for the targets mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and non-cannonical p38 in collaboration with Drs. Louis Staudt and Jonathan Ashwell (CCR). In addition we kinetically characterized inhibitors of tyrosyl-DNA phosphodiesterase-1 & 2 (Tdp1 & 2) in collaboration with Dr. Yves Pommier (CCR). MALT1 is an 824 amino acid, multi-domain protein that possesses a proteolytic paracaspase domain capable of cleaving arginine-containing substrates. MALT1 has been demonstrated to cleave multiple signaling molecules involved in NF-kappa B activation and Jun N-terminal kinase activation. In addition, MALT1 has been demonstrated to modulate T cell adhesion to fibronectin and activate caspase-8. The discovery of specific MALT1 small-molecule protease inhibitors would impact numerous aspects of cancer and immunological research. Recent research has shown that inhibition of MALT1 protease activity by RNAi and peptide inhibitors resulted in decreased survival in activated B cell-like diffuse large B-cell lymphoma cell lines, offering proof-of-principle for molecularly-targeted MALT1 therapies. In collaboration with the Staudt lab, we have developed and validated a HTS assay for inhibitors of MALT1 protease activity. The first task the PCMBS undertook was the production of active MALT1. An expression vector encoding glutathione-S-transferase (GST)-tagged, full-length human MALT1 isoform A was created. GST-MALT1 enzyme was expressed using BL21(DE3) E. coli. MALT1 was purified to near homogeneity using a combination of affinity, TEV protease cleavage/GST-readsorption, and size-exclusion chromatography. The protease activity of purified, recombinant MALT1 was monitored by cleavage of the Bcl-10 derived fluorescent peptide substrate acetyl-Leu-Arg-Ser-Arg-4-methyl-coumaryl-7-amide (Ac-LRSR-MCA). The optimized buffer for the MALT1 assay was determined to be 50 mM Tris-HCl, 1 mM DTT, 0.05% CHAPS, 0.1 mM EGTA, 0.8 M sodium citrate, pH = 7.5. Initially, the concentration of active MALT1 was determined by titration with the MALT1 irreversible inhibitor Z-VRPR-FMK. An active enzyme concentration of 497 nM was obtained, which represented 31% of the total protein. The linearity and steady-state kinetic parameters of MALT1 were then characterized. The Km of the MALT1 reaction was determined to be 103 uM. To encourage the discovery of both competitive and uncompetitive inhibitors, the substrate concentration was set to 100 uM to approximate the Km of MALT1. A combination of 100 nM MALT1 and 100 uM Ac-LRSR-MCA yielded a linear assay response through 130 minutes, and an acceptable ratio (4.9) of signal to background at 60 minutes. The addition of 0.1% SDS to the protease reaction stopped MALT1 protease activity. HTS for MALT1 inhibitory compounds is ongoing, and will include testing of MTL synthetic compound and natural product extract libraries. The goal of our collaboration with the Ashwell lab is to develop a high-throughput screen of our unique libraries to find small molecules that will specifically inhibit the alternatively activated p38 without inhibiting classically activated p38 kinase. The Ashwell lab has provided the alternatively activated form of p38 and we have established an enzymatic assay capable of sensitively detecting inhibitors of this enzyme. To ensure the specificity of these inhibitors for only the alternative pathway we have established a secondary assay using the classically activated p38 kinase. Determination of the most appropriate substrate concentration for screening is likely the most important parameter when optimizing a high-throughput biochemical assay. We will therefore carry out the assay at a substrate (ATF2-GST, [S]) concentration near the calculated Km, 300 nM. At this concentration substrate depletion is less than 25% after 60 minutes and the fractional activity should directly correlate to the anticipated Ki of the compou |