Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders.

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver.

Small-molecule IKKβ activation modulator (IKAM) targets MAP3K1 and inhibits pancreatic tumor growth.

Activation of inhibitor of nuclear factor NF-κB kinase subunit-β (IKKβ), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKβ knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKβ and its activation in cancer.

Spirocyclic dimer SpiD7 activates the unfolded protein response to selectively inhibit growth and induce apoptosis of cancer cells.

The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy.

Dimers of isatin derived α-methylene-γ-butyrolactone as potent anti-cancer agents.

The IKK-NFκB complex is a key signaling node that facilitates activation of gene expression in response to extracellular signals. The kinase IKKβ and the transcription factor RELA have been targeted by covalent modifiers that bind to surface exposed cysteine residues. A common feature in well characterized covalent modifiers of RELA and IKKβ is the Michael acceptor containing α-methylene-γ-butyrolactone functionality.

Stapling proteins in the RELA complex inhibits TNFα-induced nuclear translocation of RELA.

Tumor necrosis factor (TNF) α-induced nuclear translocation of the NF-κB subunit RELA has been implicated in several pathological conditions. Here we report the discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to inhibit TNFα-induced nuclear translocation. This is a previously unexplored strategy to inhibit TNFα-induced NF-κB activation.

Structure activity relationship (SAR) study identifies a quinoxaline urea analog that modulates IKKβ phosphorylation for pancreatic cancer therapy.

Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKKβ) as a therapeutic target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine residues (S, S) in IKKβ is a key event that drives tumor necrosis factor (TNF) α induced NF-κB mediated gene expression. Here we conducted structure activity relationship (SAR) study to improve potency and oral bioavailability of a quinoxaline analog 13-197 that was previously reported as a NFκB inhibitor for pancreatic cancer therapy.

Small molecule binding to inhibitor of nuclear factor kappa-B kinase subunit beta in an ATP non-competitive manner.

Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is a key regulator of the cannonical NF-κB pathway. IKKβ has been validated as a drug target for pathological conditions, which include chronic inflammatory diseases and cancer. Pharmacological studies revealed that chronic administration of ATP-competitive IKKβ inhibitors resulted in unexpected toxicity.

Aminopyrazole based CDK9 PROTAC sensitizes pancreatic cancer cells to venetoclax.

Cyclin-dependent kinase 9 (CDK9) is a member of the cyclin-dependent kinase (CDK) family which is involved in transcriptional regulation of several genes, including the oncogene Myc, and is a validated target for pancreatic cancer. Here we report the development of an aminopyrazole based proteolysis targeting chimera (PROTAC 2) that selectively degrades CDK9 (DC = 158 ± 6 nM). Mass spectrometry-based kinome profiling shows PROTAC 2 selectively degrades CDK9 in MiaPaCa2 cells and sensitizes them to Venetoclax mediated growth inhibition.

Correction: Characterization of CDK(5) inhibitor, 20-223 (aka CP668863) for colorectal cancer therapy.

[This corrects the article DOI: 10.18632/oncotarget.23749.

Symbiotic prodrugs (SymProDs) dual targeting of NFkappaB and CDK.

The release of an active drug from the prodrug generates a pro-fragment that typically has no biological activity and could result in adverse effects. By combining two drugs, wherein each drug acts as a pro-fragment of the other drug will eliminate the pro-fragment in the prodrug. As they are prodrugs of each other and are symbiotic, we termed these as symbiotic prodrugs (SymProDs).

CDK5 Inhibitor Downregulates Mcl-1 and Sensitizes Pancreatic Cancer Cell Lines to Navitoclax.

Developing small molecules that indirectly regulate Mcl-1 function has attracted a lot of attention in recent years. Here, we report the discovery of an aminopyrazole, 2-([1,1'-biphenyl]-4-yl)--(5-cyclobutyl-1-pyrazol-3-yl)acetamide (analog 24), which selectively inhibited cyclin-dependent kinase (CDK) 5 over CDK2 in cancer cell lines. We also show that analog 24 reduced Mcl-1 levels in a concentration-dependent manner in cancer cell lines.

Selective degradation of CDK6 by a palbociclib based PROTAC.

Development of selective kinase inhibitors that target the ATP binding site continues to be a challenge largely due to similar binding pockets. Palbociclib is a cyclin-dependent kinase inhibitor that targets the ATP binding site of CDK4 and CDK6 with similar potency. The enzymatic function associated with the kinase can be effectively probed using kinase inhibitors however the kinase-independent functions cannot.

Synthesis of aminopyrazole analogs and their evaluation as CDK inhibitors for cancer therapy.

We synthesized a library of aminopyrazole analogs to systematically explore the hydrophobic pocket adjacent to the hinge region and the solvent exposed region of cyclin dependent kinases. Structure-activity relationship studies identified an optimal substitution for the hydrophobic pocket and analog 24 as a potent and selective CDK2/5 inhibitor.

Chemical Genetic Screens Identify Kinase Inhibitor Combinations that Target Anti-Apoptotic Proteins for Cancer Therapy.

The study presented here provides a framework for the discovery of unique inhibitor combinations that target the apoptosis network for cancer therapy. A pair of doxycycline (Dox)-inducible cell lines that specifically report on the ability of an inhibitor to induce apoptosis by targeting either the Mcl-1 arm or the Bcl-2/Bcl-xL/Bcl-w arm were used. Cell-based assays were optimized for high throughput screening (HTS) with caspase 3/7 as a read out.

Characterization of CDK(5) inhibitor, 20-223 (aka CP668863) for colorectal cancer therapy.

Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for patients suffering from CRC, none of which focus on the cell signaling mechanisms controlled by the popular kinase family, cyclin dependent kinases (CDKs). Here we evaluate a Pfizer developed compound, CP668863, that inhibits cyclin-dependent kinase 5 (CDK5) in neurodegenerative disorders.

Characterization of Promiscuous Binding of Phosphor Ligands to Breast-Cancer-Gene 1 (BRCA1) C-Terminal (BRCT): Molecular Dynamics, Free Energy, Entropy and Inhibitor Design.

Inhibition of the protein-protein interaction (PPI) mediated by breast-cancer-gene 1 C-terminal (BRCT) is an attractive strategy to sensitize breast and ovarian cancers to chemotherapeutic agents that induce DNA damage. Such inhibitors could also be used for studies to understand the role of this PPI in DNA damage response. However, design of BRCT inhibitors is challenging because of the inherent flexibility associated with this domain.

Isatin Derived Spirocyclic Analogues with α-Methylene-γ-butyrolactone as Anticancer Agents: A Structure-Activity Relationship Study.

Design, synthesis, and evaluation of α-methylene-γ-butyrolactone analogues and their evaluation as anticancer agents is described. SAR identified a spirocyclic analogue 19 that inhibited TNFα-induced NF-κB activity, cancer cell growth and tumor growth in an ovarian cancer model. A second iteration of synthesis and screening identified 29 which inhibited cancer cell growth with low-μM potency.

The paradox of conformational constraint in the design of Cbl(TKB)-binding peptides.

Solving the crystal structure of Cbl(TKB) in complex with a pentapeptide, pYTPEP, revealed that the PEP region adopted a poly-L-proline type II (PPII) helix. An unnatural amino acid termed a proline-templated glutamic acid (ptE) that constrained both the backbone and sidechain to the bound conformation was synthesized and incorporated into the pYTPXP peptide. We estimated imposing structural constraints onto the backbone and sidechain of the peptide and preorganize it to the bound conformation in solution will yield nearly an order of magnitude improvement in activity.

Peptide truncation leads to a twist and an unusual increase in affinity for casitas B-lineage lymphoma tyrosine kinase binding domain.

We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape.

Computational and experimental studies of the interaction between phospho-peptides and the C-terminal domain of BRCA1.

The C-terminal domain of BRCA1(BRCT) is involved in the DNA repair pathway by recognizing the pSXXF motif in interacting proteins. It has been reported that short peptides containing this motif bind to BRCA1(BRCT) in the micromolar range with high specificity. In this work, the binding of pSXXF peptides has been studied computationally and experimentally in order to characterize their interaction with BRCA1(BRCT).

Exploiting the P-1 pocket of BRCT domains toward a structure guided inhibitor design.

Breast cancer gene 1 carboxy terminus (BRCT) domains are found in a number of proteins that are important for DNA damage response (DDR). The BRCT domains bind phosphorylated proteins and these protein-protein interactions are essential for DDR and DNA repair. High affinity domain specific inhibitors are needed to facilitate the dissection of the protein-protein interactions in the DDR signaling.

Structure-activity relationship studies to probe the phosphoprotein binding site on the carboxy terminal domains of the breast cancer susceptibility gene 1.

Carboxy terminal BRCT domains of the breast cancer susceptibility gene 1 (BRCA1) bind to phosphorylated proteins through a pSXXF consensus recognition motif. We report a systematic structure-activity relationship study that maps the BRCT(BRCA1)-pSXXF binding interface, leading to identification of peptides with nanomolar binding affinities comparable to those of the previously reported 13-mer peptides and providing a clear description of the pSXXF-BRCT interface, which is essential for developing small molecule inhibitors via the peptidomimetic approach.

Structural characterization of BRCT-tetrapeptide binding interactions.

BRCT(BRCA1) plays a major role in DNA repair pathway, and does so by recognizing the conserved sequence pSXXF in its target proteins. Remarkably, tetrapeptides containing pSXXF motif bind with high specificity and micromolar affinity. Here, we have characterized the binding interactions of pSXXF tetrapeptides using NMR spectroscopy and calorimetry.