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

Activation of inhibitor of nuclear factor NF-κB kinase subunit-ß (IKKß), 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 IKKß 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 IKKß and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKß is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKß in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKß inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKß but inhibited the IKKß kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKß phosphorylation was inhibited by 39-100, thus we termed it IKKß activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKß levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKß activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

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 IKKß 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 IKKß is the Michael acceptor containing α-methylene-γ-butyrolactone functionality. Through synthesis and evaluation of a focused set of α-methylene-γ-butyrolactone containing spirocyclic dimers (SpiDs) we identified SpiD3 as an anticancer agent with low nanomolar potency. Using cell-free and cell-based studies we show that SpiD3 is a covalent modifier that generates stable RELA containing high molecular weight complexes. SpiD3 inhibits TNFα-induced IκBα phosphorylation resulting in the blockade of RELA nuclear translocation. SpiD3 induces apoptosis, inhibits colony formation and migration of cancer cells. The NCI-60 cell line screen revealed that SpiD3 potently inhibits growth of leukemia cell lines, making it a suitable pre-therapeutic lead for hematological malignancies.

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 (DC50 = 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.

Synthesis and SAR studies of novel 1,2,4-oxadiazole-sulfonamide based compounds as potential anticancer agents for colorectal cancer therapy.

A diverse series of 1,2,4-oxadiazoles based substituted compounds were designed, synthesized and evaluated as anticancer agents targeting carbonic anhydrase IX (CAIX). Initial structure-activity analysis suggested that the thiazole/thiophene-sulfonamide conjugates of 1,2,4-oxadiazoles exhibited potent anticancer activities with low µM potencies. Compound OX12 exhibited antiproliferative activity (IC50 = 11.1 µM) along with appreciable inhibition potential for tumor-associated CAIX (IC50 = 4.23 µM) isoform. Therefore, OX12 was structurally optimized and its SAR oriented derivatives (OX17-27) were synthesized and evaluated. This iteration resulted in compound OX27 with an almost two-fold increase in antiproliferative effect (IC50 = 6.0 µM) comparable to the clinical drug doxorubicin and significantly higher potency against CAIX (IC50 = 0.74 µM). Additionally, OX27 treatment decreases the expression of CAIX, induces apoptosis and ROS production, inhibited colony formation and migration of colon cancer cells. Our studies provide preclinical rational for the further optimization of identified OX27 as a suitable lead for the possible treatment of CRC.

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)-N-(5-cyclobutyl-1H-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. Using a panel of doxycycline inducible cell lines, we show that CDK5 inhibitor 24 selectively modulates Mcl-1 function while the CDK4/6 inhibitor 6-acetyl-8-cyclopentyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-day]pyrimidin-7(8H)-one does not. Previous studies using RNA interference and CRISPR showed that concurrent elimination of Bcl-xL and Mcl-1 resulted in induction of apoptosis. In pancreatic cancer cell lines, we show that either CDK5 knockdown or expression of a dominant negative CDK5 results in synergistic induction of apoptosis. Moreover, concurrent pharmacological perturbation of Mcl-1 and Bcl-xL in pancreatic cancer cell lines using a CDK5 inhibitor analog 24 that reduced Mcl-1 levels and 4-(4-{[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl] benzamide (navitoclax), a Bcl-2/Bcl-xL/Bcl-w inhibitor, resulted in synergistic inhibition of cell growth and induction of apoptosis. In conclusion, we demonstrate targeting CDK5 will sensitize pancreatic cancers to Bcl-2 protein inhibitors. SIGNIFICANCE STATEMENT: Mcl-1 is stabilized by CDK5-mediated phosphorylation in pancreatic ductal adenocarcinoma, resulting in the deregulation of the apoptotic pathway. Thus, genetic or pharmacological targeting of CDK5 sensitizes pancreatic cancers to Bcl-2 inhibitors, such as navitoclax.

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. Herein, we report a palbociclib based PROTAC that selectively degrades CDK6 while sparing the homolog CDK4. We used competition studies to characterize the binding and mechanism of CDK6 degradation.

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. HTS with a 355-member kinase inhibitor library and the panel of Dox-inducible cell lines revealed that cyclin dependent kinase (CDK) inhibitors induced apoptosis by targeting the Mcl-1 arm, whereas PI3K inhibitors induced apoptosis by targeting the Bcl-2/Bcl-xL/Bcl-w arm. Validation studies identified unique combinations that synergistically inhibited growth and induced apoptosis in a panel of cancer cell lines. Since these inhibitors have been or are currently in clinical trials as single agents, the combinations can be rapidly translated to the clinics.