• Inhibition of TLR2 Signaling by Small Molecule Inhibitors Targeting a Pocket Within the TLR2 TIR Domain

      Mistry, Pragnesh Dilipkumar; Vogel, Stefanie N. (2015)
      Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll-Interleukin-1 receptor resistance (TIR) domains. For all TLRs, except TLR3, recruitment of the adapter, MyD88, to the TIR domains results in downstream signaling that culminates in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate diseases caused by TLR-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a "pocket" adjacent to the highly conserved P681 and G682 residues of the BB loop. Using "Computer-Aided Drug Design" (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this "BB loop pocket" and, potentially, disrupt TLR2 signaling. In silico screening identified 149 lead compounds and 20 FDA-approved drugs based on their predicted ability to bind in the BB loop pocket. These were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. In this screen, C16H15NO4 ("C29") was identified as a potential TLR2 inhibitor. C29, and a derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages induced by synthetic and bacterial TLR2 agonists. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6 signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle for this novel CADD approach for the identification of inhibitors of TLR signaling.
    • Structure-based optimization of small molecule transcription therapy targeting the BCL6 oncoprotein

      Tsourounis, Marilyn; Coop, Andrew (2009)
      BCL6, a member of the BTB/Zinc finger family of proteins is a transcriptional repressor that is constitutively expressed in over 50% of patients with Diffuse Large B Cell Lymphomas (DLBCL). BCL6 is responsible for the repression of at least 14 different genes linked by roles in B cell activation, differentiation, inflammation, and cell cycle control. Prior to cell differentiation, normal BCL6 encourages apoptosis by repressing expression of anti-apoptotic BCL6 target genes in the germinal center. Specific mutations and translocations in the 5' regulatory region of the BCL6 gene have been implicated in the constitutive expression of BCL6. This results in the aberrant replication of B cells that can escape the germinal center and lead to the formation of lymphomas. Preliminary proof of principle experiments have illustrated that an interaction between the BTB domain of BCL6 and unique regulatory sequences of its critical corepressors NCoR and SMRT are responsible for the mechanism of transcriptional repression of BCL6 at a "lateral groove" motif. Peptides that block the BCL6-SMRT interface kill lymphoma cells in vitro and in vivo, without causing toxicity to normal tissues. Small molecule inhibitors based on the 3D BCL6-SMRT interface were discovered via a target based drug design approach utilizing computer aided chemical database screening. Lead compounds displaying the desired biological activity in biochemical and functional assays were identified and selected for chemical derivation. Over 70 compounds have been developed and synthesized and are currently being evaluated for BCL6 inhibitory activity, as a part of our successive chemical optimization process to improve binding affinity, selectivity, and solubility. Preliminary in vitro studies of our first generations of indolylidene inhibitor compounds have demonstrated that an N-carboxylic acid and nature of the central heterocyclic ring are significant for activity. These results are consistent with CADD results determination of an energetically favorable interaction between the carboxylic charged moieties of lead compounds identified from primary database screens and key Arginine and Histidine residues in the putative binding site of the BCl6 BTB domain. Other CADD identified lead candidates possessing a diarylheterocyclic based scaffold were selected for further optimization to generate a chemically diverse library of molecules for SAR investigation of BTB lateral groove blockade and are presented. As continuation of this work, the development of small molecule inhibitors to target a secondary binding site of the BTB domain is described. Our structure-based strategy for introducing chemical modifications to our existing small molecule inhibitors along with proposed synthetic methodology to develop targets achieving this unique biological profile are reported.