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

Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKKß) as a therapeutic target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine residues (S177, S181) in IKKß 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. The SAR led to the identification of a novel quinoxaline urea analog 84 that reduced the levels of p-IKKß in dose- and time-dependent studies. When compared to 13-197, analog 84 was ∼2.5-fold more potent in TNFα-induced NFκB inhibition and ∼4-fold more potent in inhibiting pancreatic cancer cell growth. Analog 84 exhibited ∼4.3-fold greater exposure (AUC0-∞) resulting in ∼5.7-fold increase in oral bioavailability (%F) when compared to 13-197. Importantly, oral administration of 84 by itself and in combination of gemcitabine reduced p-IKKß levels and inhibited pancreatic tumor growth in a xenograft model.

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.

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.

Recent Advances in Cancer Drug Development: Targeting Induced Myeloid Cell Leukemia-1 (Mcl-1) Differentiation Protein.

BACKGROUND: Anti-apoptotic members of the Bcl-2 family of proteins are upregulated in a majority of cancers and are potential therapeutic targets. Fragment-based design led to the development of clinical candidates that target Bcl-xL/Bcl-2. Although these BclxL/ Bcl-2 inhibitors showed promise in pre-clinical studies, resistance to several Bcl-xL inhibitors was observed, when used alone. This is attributed to the over-expression of Mcl-1, another member of the Bcl-2 family of proteins. Indeed, Mcl-1 is highly amplified in numerous cancers, suggesting that it may contribute to malignant cell growth and evasion of apoptosis. Therefore, significant efforts have been made toward the development of direct Mcl-1 inhibitors for cancer therapy. METHODS: Following an extensive search of peer-reviewed articles on the development of Mcl-1-selective inhibitors, the literature retrieved is chronologically arranged and discussed in this review article. RESULTS: We have included 147 articles in this review; including articles that describe the development of stapled peptides with improved binding affinity as Mcl-1-selective BH3 mimetics, those describing fragment-based and structure-based design of small molecule Mcl-1 inhibitors by various research groups, and those detailing the use of natural products and their derivatives as potential Mcl-1 inhibitors. CONCLUSION: The therapeutic potential of targeting the Mcl-1 protein for cancer drug discovery is vast. Stapling BH3 peptides, as well as the development of small molecule inhibitors as BH3 mimetics, are viable strategies to develop selective Mcl-1 inhibitors. With no clinically approved candidate in hand, additional modes of perturbing the biological function of this protein will aid drug discovery efforts.

Development of 1-((1,4-trans)-4-Aryloxycyclohexyl)-3-arylurea Activators of Heme-Regulated Inhibitor as Selective Activators of the Eukaryotic Initiation Factor 2 Alpha (eIF2α) Phosphorylation Arm of the Integrated Endoplasmic Reticulum Stress Response.

Heme-regulated inhibitor (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, adaptation to stress, and hemoglobin disorders. HRI phosphorylates eIF2α, which couples cellular signals, including endoplasmic reticulum (ER) stress, to translation. We previously identified 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific activators of HRI that trigger the eIF2α phosphorylation arm of ER stress response as molecular probes for studying HRI biology and its potential as a druggable target. To develop drug-like cHAUs needed for in vivo studies, we undertook bioassay-guided structure-activity relationship studies and tested them in the surrogate eIF2α phosphorylation and cell proliferation assays. We further evaluated some of these cHAUs in endogenous eIF2α phosphorylation and in the expression of the transcription factor C/EBP homologous protein (CHOP) and its mRNA, demonstrating significantly improved solubility and/or potencies. These cHAUs are excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

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.

Developing Polyamine-Based Peptide Amphiphiles with Tunable Morphology and Physicochemical Properties.

The ability to tune supramolecular properties such as size, morphology, or metabolic stability is of paramount importance in the field of supramolecular chemistry. Peptide amphiphiles (PAs) are a family of functional self-assembling biomaterials that have garnered widespread attention due to their broad applicability in medicine. PAs are generally comprised of an amino acid sequence connected to lipid tail(s) allowing them to self-assemble into supramolecular structures with diverse morphologies. Herein, this study describes the synthesis of a new class of polyamine-based "hybrid" PAs (PPAs) as novel self-assembling systems. The described molecules possess diverse polyamine head groups with the goal of tuning physicochemical properties. The findings indicate that small changes in the polyamine head groups result in altered PPA morphologies (nanofibers, micelles, nanoworms). The PPAs present a wide range of physicochemical characteristics, show superior resistance to aggregation, a diverse metabolic profile, and varied assembling kinetics. Most of the PPAs do not show toxicity in the human cells lines evaluated. The PPAs described herein hold promising potential as a safe and nontoxic option for drug delivery, targeting, and tissue engineering applications.

Discovery and characterization of small molecule Rac1 inhibitors.

Aberrant activation of Rho GTPase Rac1 has been observed in various tumor types, including pancreatic cancer. Rac1 activates multiple signaling pathways that lead to uncontrolled proliferation, invasion and metastasis. Thus, inhibition of Rac1 activity is a viable therapeutic strategy for proliferative disorders such as cancer. Here we identified small molecule inhibitors that target the nucleotide-binding site of Rac1 through in silico screening. Follow up in vitro studies demonstrated that two compounds blocked active Rac1 from binding to its effector PAK1. Fluorescence polarization studies indicate that these compounds target the nucleotide-binding site of Rac1. In cells, both compounds blocked Rac1 binding to its effector PAK1 following EGF-induced Rac1 activation in a dose-dependent manner, while showing no inhibition of the closely related Cdc42 and RhoA activity. Furthermore, functional studies indicate that both compounds reduced cell proliferation and migration in a dose-dependent manner in multiple pancreatic cancer cell lines. Additionally, the two compounds suppressed the clonogenic survival of pancreatic cancer cells, while they had no effect on the survival of normal pancreatic ductal cells. These compounds do not share the core structure of the known Rac1 inhibitors and could serve as additional lead compounds to target pancreatic cancers with high Rac1 activity.

Cyclin Dependent Kinase 9 Inhibitors for Cancer Therapy.

Cyclin dependent kinase (CDK) inhibitors have been the topic of intense research for nearly 2 decades due to their widely varied and critical functions within the cell. Recently CDK9 has emerged as a druggable target for the development of cancer therapeutics. CDK9 plays a crucial role in transcription regulation; specifically, CDK9 mediated transcriptional regulation of short-lived antiapoptotic proteins is critical for the survival of transformed cells. Focused chemical libraries based on a plethora of scaffolds have resulted in mixed success with regard to the development of selective CDK9 inhibitors. Here we review the regulation of CDK9, its cellular functions, and common core structures used to target CDK9, along with their selectivity profile and efficacy in vitro and in vivo.

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. Our data suggest that an isatin-derived spirocyclic α-methylene-γ-butyrolactone is a suitable core for optimization to identify novel anticancer agents.