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 (Tdp1) 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 compound. At this concentration, we believe that we can reasonably collect 500,000 data points at an acceptable substrate cost. Idiopathic activation of the immune system is a central mediator of autoimmune disorders like rheumatoid arthritis and systemic lupus erythematous. Autoimmune disorders are already highly prevalent and are predicted to increase as the global population ages. Therefore, understanding the etiology of these diseases and developing novel therapeutics to combat them is critically important. To this end we are collaborating with Dr. Jonathan Ashwell (LICB), who has identified a novel mechanism by which the immune system can become inappropriately activated. One mechanism of T-cell activation is through the engagement of the T-cell receptor (TCR), which initiates an intracellular kinase signaling cascade ultimately resulting in the production of pro-inflammatory cytokines and the induction of an inflammatory response. The canonical class of kinases that transduces the TCR activation signal into an inflammatory response is that of the Mitogen Activated Protein Kinase (MAPK) family, of which the p38 kinase is the pre-eminent member. Prior to the recent work of the Ashwell lab it was understood that p38 activation was the result of a signaling cascade dependent on a phosphorelay through a series of upstream MAPK family members (MKK3/4/6). This ultimately results in the complete activation of p38 through the dual phosphorylation of its activation loop at threonine 180 (T180) and tyrosine 182 (Y182). The Ashwell lab has now demonstrated that TCR engagement can mediate p38 activation without the classical signaling cascade through the activation of a TCR proximal kinase, ZAP70, which results in the novel phosphorylation of p38 at tyrosine 323 (Y323). Unlike the classically activated p38, phosphorylation at Y323 imbues p38 with the ability to autophosphorylate its own activation loop, thus activating itself in a feed forward signaling cascade. It has been demonstrated that this phenomenon is T-cell specific and is normally opposed in T-cells by an inhibitory scaffolding protein, GADD45-alpha. In the absence of GADD45-alpha mediated inhibition the alternatively activated p38 initiates a pro-inflammatory cascade and subsequent T-cell dependent inflammation ensues. The central goal of our collaboration with the Ashwell lab is to bring to bear our expertise in developing high-throughput screens and our unique libraries of both pure compounds and natural product extracts to find small molecules that will specifically inhibit the alternatively activated p38 (phosphorylated at Y323) but will not inhibit the classically activated p38 kinase (unphosphorylated at Y323). To accomplish this goal, 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 (phosphorylated at T180 and Y182). Only those inhibitors that specifically inhibit the alternatively activated pathway will be considered hits for the purposes of this screen. Additionally, the Ashwell lab will test these hits for their ability to specifically inhibit only the alternative pathway in several T-cell models. We believe we have the requisite sensitivity in our primary screen and the commensurate specificity in our secondary assays to probe the full chemical diversity of our compound libraries for inhibitors of this pathway.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011471-02
Application #
8763550
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$814,972
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code