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.

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. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics.

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.

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). To test this idea, we generated SymProDs using NFκB inhibitors that contain the reactive α-methylene-γ-butyrolactone moiety and CDK inhibitors with solvent exposed secondary nitrogen atoms. We show that secondary amine prodrugs of α-methylene-γ-butyrolactone containing NFκB inhibitors undergo slow release over a 72 hr period. Using an alkyne-tagged secondary amine prodrug of α-methylene-γ-butyrolactone containing NFκB inhibitor, we demonstrate target engagement. The NFκB-CDK SymProDs were ~20- to 200-fold less active against the corresponding CDK inhibitors in in vitro CDK kinase assays. Growth inhibition studies in a panel of ovarian cancer cell lines revealed potency trends of the SymProDs mirrored those of the single treatments suggesting their dissociation in cells. In conclusion, our results suggest that SymProDs offer a productive path forward for advancing compounds with reactive functionality and can be used as dual targeting agents.

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.

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. CDK5 has been implicated in a number of cancers, most recently as an oncogene in colorectal cancers. Our lab synthesized and characterized CP668863 - now called 20-223. In our established colorectal cancer xenograft model, 20-223 reduced tumor growth and tumor weight indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. In our hands, in vitro 20-223 is most potent against CDK2 and CDK5. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core and we used it to benchmark the 20-223 potency. In CDK5 and CDK2 kinase assays, 20-223 was ∼3.5-fold and ∼65.3-fold more potent than known clinically used CDK inhibitor, AT7519, respectively. Cell-based studies examining phosphorylation of downstream substrates revealed 20-223 inhibits the kinase activity of CDK5 and CDK2 in multiple CRC cell lines. Consistent with CDK5 inhibition, 20-223 inhibited migration of CRC cells in a wound-healing assay. Profiling a panel of CRC cell lines for growth inhibitory effects showed that 20-223 has nanomolar potency across multiple CRC cell lines and was on an average >2-fold more potent than AT7519. Cell cycle analyses in CRC cells revealed that 20-223 phenocopied the effects associated with AT7519. Collectively, these findings suggest that 20-223 exerts anti-tumor effects against CRC by targeting CDK 2/5 and inducing cell cycle arrest. Our studies also indicate that 20-223 is a suitable lead compound for colorectal cancer therapy.

Chemically induced degradation of CDK9 by a proteolysis targeting chimera (PROTAC).

Cyclin-dependent kinase 9 (CDK9), a member of the cyclin-dependent protein kinase (CDK) family, is involved in transcriptional elongation of several target genes. CDK9 is ubiquitously expressed and has been shown to contribute to a variety of malignancies such as pancreatic, prostate and breast cancers. Here we report the development of a heterobifunctional small molecule proteolysis targeting chimera (PROTAC) capable of cereblon (CRBN) mediated proteasomal degradation of CDK9. In HCT116 cells, it selectively degrades CDK9 while sparing other CDK family members. This is the first example of a PROTAC that selectively degrades CDK9.

Long-circulatory nanoparticles for gemcitabine delivery: Development and investigation of pharmacokinetics and in-vivo anticancer efficacy.

The anticancer potential of gemcitabine, a nucleoside analog, is compromised due to the enzymatic degradation into inactive form leading to the short half-life in systemic circulation. Novel delivery strategies are required to improve therapeutic efficacy of this potential drug. Monomethoxy polyethylene glycol amine-polylactide-co-glycolide (mPEG-PLGA) co-polymer was synthesized and characterized by FTIR and (1)H NMR. Gemcitabine loaded mPEG-PLGA nanoparticles (NPs) were developed and investigated for pharmacokinetic profile and in vivo anticancer activity. The mPEG-PLGA NPs (size: 267±10nm, zeta potential: -17.5±0.2mV) exhibited sustained drug release profile and were found to be compatible with blood. The mPEG-PLGA NPs were able to evade the uptake by macrophages (i.e. THP-1 and J774A) by reducing the adsorption of proteins on the surface of NPs. The enhanced cellular uptake and cell cytotoxicity was observed by mPEG-PLGA NPs in MiaPaCa-2 and MCF-7 cells. The half-life of gemcitabine in mPEG-PLGA NPs was remarkably enhanced (19 folds) than native gemcitabine. Further, the pharmacokinetic modulation of gemcitabine using mPEG-PLGA-NPs was translated in improved anticancer efficacy as compared to native gemcitabine in Ehrlich ascites bearing Balb-c mice. The results demonstrated the potential of long-circulatory nanoparticles in improving the pharmacokinetic profile and in-turn the anticancer efficacy of gemcitabine.

Development and evaluation of paclitaxel loaded PLGA:poloxamer blend nanoparticles for cancer chemotherapy.

This investigation described the development of novel PLGA:poloxamer blend nanoparticles for intravenous administration of paclitaxel in order to limit the cremophor-associated adverse effects. The developed formulation was well-characterized using various techniques including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. The nanoparticles had an average particle size around 180nm and zeta potential of -22.7mV. The in vitro release study of nanoparticles exhibited biphasic release pattern. The non-hemolytic potential of the nanoparticles indicated the suitability of the developed formulation for intravenous administration. The PLGA:poloxamer blend nanoparticles showed significantly improved cytotoxicity in cell lines (MCF-7 and Colo-205), as compared to free drug. Further, the developed formulation was stable under the accelerated storage conditions. In conclusion, the results indicated that the developed polymeric formulation is a novel and potential alternative for the paclitaxel delivery.

Synthesis, characterization and mechanistic-insight into the anti-proliferative potential of PLGA-gemcitabine conjugate.

Gemcitabine, a nucleoside analogue, is used in the treatment of various solid tumors, however, its efficacy is limited by rapid metabolism by cytidine deaminase and fast kidney excretion. In this study, a polymeric conjugate of gemcitabine was prepared by covalent coupling with poly(lactic-co-glycolic) acid (PLGA), in order to improve anticancer efficacy of the drug. The prepared conjugate was characterized by various analytical techniques including FTIR, NMR and mass spectroscopic analysis. The stability study indicated that the polymeric conjugate was more stable in plasma as compared to native gemcitabine. Further, in vitro cytotoxicity determined in a panel of cell lines including pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116), indicated that the cytotoxic activity of gemcitabine was retained following conjugation with polymeric carrier. In the nucleoside transportation inhibition assay, it was found that the prepared conjugate was not dependent on nucleoside transporter for entering into the cells and this, in turn, reflecting potential implication of this conjugate in the therapy of transporter- deficient resistance cancer. Further, the cell cycle analysis showed that the sub-G1 (G0) apoptotic population was 46.6% and 60.6% for gemcitabine and PLGA gemcitabine conjugate, respectively. The conjugate produced remarkable decrease in mitochondrial membrane potential, a marker of apoptosis. In addition, there was a marked increase in PARP cleavage and P-H2AX expression with PLGA gemcitabine conjugate as compared to native gemcitabine indicating improved apoptotic activity. The findings demonstrated the potential of PLGA gemcitabine conjugate to improve clinical outcome of gemcitabine based chemotherapy of cancer.

Synthesis, antiproliferative and apoptosis-inducing activity of thiazolo[5,4-d]pyrimidines.

Thiazolo[5,4-d]pyrimidines are important class of heterocyclic compounds possessing diverse range of biological activities. Herein, we report an efficient synthesis of thiazolo[5,4-d]pyrimidines using recyclable KF/alumina catalyst. The reaction of 4,6-dichloro-5-aminopyrimidine with isothiocyanates in presence of 20 mol% KF/alumina produced thiazolo[5,4-d]pyrimidines in excellent yields without any chromatographic purifications. The method is operationally simple, fast and the catalyst can be reused without any significant loss of activity. These compounds were tested for antiproliferative activity in a panel of 8 cancer cell lines, including lung (NCI-H322 and A549), epidermal (A431), glioblastoma (T98G), pancreatic (MIAPaCa-2), prostate (PC-3), human leukemia (HL-60) and breast (T47D) cells. The N,N'-diethylamino-substituted analog, 2-(4-chlorophenylamino)-7-diethylamino-thiazolo[5,4-d]pyrimidine 4k showed antiproliferative activity in lung (NCI-H322 and A549), epidermal (A431) and glioblastoma (T98G) cancer cell lines with IC50 values of 7.1, 1.4, 3.1 and 3.4 µM, respectively. The morpholine substituted analog 4a displayed activity in HL-60 cells with IC50 value of 8 µM. The compound 4k showed induction of apoptosis in A549 cells at 10 µM, as indicated by the increase in the sub-G1 population. The nuclear morphology of A549 cells after treatment with 4k was also investigated. Similarly, the morpholine substituted analog 4a induced apoptosis in HL-60 cells at 20 µM. The effect of compound 4a on mitochondrial potential loss in HL-60 cells was also studied. Further, western blotting of 4a and 4k showed cleavage of PARP-1 and procaspase-3 inhibition which confirms their apoptosis-inducing activity.