This project resulted in the development high throughput screens for the targets mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and Polo-like kinase 1 (Plk1) in collaboration with Drs. Louis Staudt and Kyung Lee (CCR). In addition we kinetically characterized inhibitors of tyrosyl-DNA phosphodiesterase-1 (Tdp1) in collaboration with Dr. Yves Pommier (CCR). Plk1 belongs to the polo-like kinase family of Ser/Thr protein kinases, and similarly to the cyclin dependent kinases, plays an important role in cellular proliferation. It is overexpressed in 80% of human cancers. In contrast, the two most closely related kinases, Plk2 and Plk3, appear to play a role in checkpoint-mediated cell cycle arrest to ensure genetic stability. Specific inhibition of Plk1, but not Plk2 or Plk3, is critical for anti-Plk cancer therapy. The Plk proteins have two functionally crucial target sites, a kinase domain that is closely related to several other protein kinases and the unique polo-box domain (PBD), which has been shown to confer specificity. As such, compounds that target the kinase domain may be less specific for Plk1, whereas those that target the PBD domain may be more specific. The goal of this project is to identify inhibitors of Plk1. In collaboration with Dr. Kyung Lee (LM) we have designed a competitive binding assay for natural product extracts that inhibit binding between a pT78 peptide and the PBD of Plk1. An ELISA assay was modified to 384-well plates and screened against MTL pure compound libraries as well as the MTL pre-fractionated natural product extract library. The assay begins with a coating step, where a biotinylated pT78 peptide (a 9-mer PBIP sequence) is added to the bottom of a streptavidin-coated black 384-well plate. The assay progresses to a binding step where full-length GST-Plk1 is added (alone and in the presence of test samples) to the surface-bound pT78 peptide. The level of bound Plk1 is measured by ELISA using a Plk1-specific primary antibody visualized by colorimetric detection. A total of 97,504 pre-fractionated natural product extract samples were screened resulting in 1573 initial hits. These were filtered to prioritize 440 extracts with a Z score >4 for confirmation assays (0.45% initial hit rate). The confirmation assays resulted in 185 natural product extract fractions, which were then prioritized according to NPCS criteria and tested in dose response assays. Dose response results identified 72 fractions (0.07% final hit rate) for further bioassay-guided fractionation. Out of the 72 lead fraction samples, 24 hits had IC50 <20 mg/ml and 48 hits had IC50 values between 20-50 mg/ml. 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. A major class of immune cells that mediate the immune response is the T-cell. 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 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 classically activated p38, phosphorylation at Y323 imbues p38 with the ability to autophosphorylate its own activation loop. 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.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.
