Regulated Induced Proximity Targeting Chimeras (RIPTACs): a Novel Heterobifunctional Small Molecule Therapeutic Strategy for Killing Cancer Cells Selectively.

While specific cell signaling pathway inhibitors have yielded great success in oncology, directly triggering cancer cell death is one of the great drug discovery challenges facing biomedical research in the era of precision oncology. Attempts to eradicate cancer cells expressing unique target proteins, such as antibody-drug conjugates (ADCs), T-cell engaging therapies, and radiopharmaceuticals have been successful in the clinic, but they are limited by the number of targets given the inability to target intracellular proteins. More recently, heterobifunctional small molecules such as Proteolysis Targeting Chimera (PROTACs) have paved the way for protein proximity inducing therapeutic modalities. Here, we describe a proof-of-concept study using novel heterobifunctional small molecules called Regulated Induced Proximity Targeting Chimeras or RIPTACs, which elicit a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting cooperative protein:protein interaction (PPI) abrogates the function of the essential protein, thus leading to cell death selectively in cells expressing the target protein. This approach not only opens new target space by leveraging differentially expressed intracellular proteins but also has the advantage of not requiring the target to be a driver of disease. Thus, RIPTACs can address non-target mechanisms of resistance given that cell killing is driven by inactivation of the essential protein. Using the HaloTag7-FKBP model system as a target protein, we describe RIPTACs that incorporate a covalent or non-covalent target ligand connected via a linker to effector ligands such as JQ1 (BRD4), BI2536 (PLK1), or multi-CDK inhibitors such as TMX3013 or dinaciclib. We show that these RIPTACs exhibit positive co-operativity, accumulate selectively in cells expressing HaloTag7-FKBP, form stable target:RIPTAC:effector trimers in cells, and induce an anti-proliferative response in target-expressing cells. We propose that RIPTACs are a novel heterobifunctional therapeutic modality to treat cancers that are known to selectively express a specific intracellular protein.

HIPK2 directs cell type-specific regulation of STAT3 transcriptional activity in Th17 cell differentiation.

SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma.

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

Selective PP2A Enhancement through Biased Heterotrimer Stabilization.

Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

Bromodomain biology and drug discovery.

The bromodomain (BrD) is a conserved structural module found in chromatin- and transcription-associated proteins that acts as the primary reader for acetylated lysine residues. This basic activity endows BrD proteins with versatile functions in the regulation of protein-protein interactions mediating chromatin-templated gene transcription, DNA recombination, replication and repair. Consequently, BrD proteins are involved in the pathogenesis of numerous human diseases. In this Review, we highlight our current understanding of BrD biology, and discuss the latest development of small-molecule inhibitors targeting BrDs as emerging epigenetic therapies for cancer and inflammatory disorders.

Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.

Pyrimido[4,5-b]indole derivatives and use thereof in the expansion of hematopoietic stem cells US2015011543 (a1): a patent evaluation.

INTRODUCTION: There is an unmet need of strategies for ex-vivo expansion of hematopoetic stem cells (HSCs) without loss of their primitive nature or stemness. We evaluate here a patent that attempts to address this need via key small molecules 1 and 40 that possess a pyrimido[4,5-b]indole core. Areas covered: (i) Discussion on literature reports of diverse strategies for ex-vivo expansion of stem cells. (ii) Synthetic scheme to 1, and general synthetic schemes for compounds 1-55 reported in the patent application. (iii) Analysis of the in vitro biological data for 1 and 40. Highlight here is: 1 and 40 when used in combination with StemReginin1 (SR1), an established aryl hydrocarbon receptor antagonist known for ex-vivo HSC expansion, demonstrate better HSC expansion relative to SR1 alone. (iv) Analysis of the in vivo biological data for 1 and 40. Expert opinion: Compelling evidence on the molecular mechanism of action of 1 and 40 is not provided making it difficult to optimize this series. It is suggested here that combining these molecules with homing molecules will possibly improve overall engraftment time and hematopoietic recovery. The numerous literature reports and biological data indicates that these pyrimido[4,5-b]indole derivatives are promising candidates for the development of potential therapies for hematopoietic ailments.

Chemical modulators for epigenome reader domains as emerging epigenetic therapies for cancer and inflammation.

Site-specific lysine acetylation and methylation on histones are critical post-translational modifications (PTMs) that govern ordered gene transcription in chromatin. Mis-regulation of these histone PTM-mediated processes has been shown to be associated with human diseases. Since the 2010 landmark reports of small molecules (+)-JQ1 and I-BET762 that target the acetyl-lysine 'reader' Bromodomain and Extra Terminal domain (BET) proteins, there have been relentless efforts to develop epigenetic therapy with small molecules to modulate molecular interactions of epigenome reader domain proteins with PTMs. In addition to BET, the other emerging targets include non-BET acetyl-lysine and methyl-lysine reader domains. This review covers the key chemical modulators of the aforementioned epigenome reader proteins.

Design, synthesis, and structure-activity relationships of pyrimido[4,5-b]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells.

To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2-8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2-8. Compounds 2 and 6 had two-digit nanomolar potency (IC50) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the ßIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles.

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

Synthesis and evaluation of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents.

In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.

AG311, a small molecule inhibitor of complex I and hypoxia-induced HIF-1α stabilization.

Cancer cells have a unique metabolic profile and mitochondria have been shown to play an important role in chemoresistance, tumor progression and metastases. This unique profile can be exploited by mitochondrial-targeted anticancer therapies. A small anticancer molecule, AG311, was previously shown to possess anticancer and antimetastatic activity in two cancer mouse models and to induce mitochondrial depolarization. This study defines the molecular effects of AG311 on the mitochondria to elucidate its observed efficacy. AG311 was found to competitively inhibit complex I activity at the ubiquinone-binding site. Complex I as a target for AG311 was further established by measuring oxygen consumption rate in tumor tissue isolated from AG311-treated mice. Cotreatment of cells and animals with AG311 and dichloroacetate, a pyruvate dehydrogenase kinase inhibitor that increases oxidative metabolism, resulted in synergistic cell kill and reduced tumor growth. The inhibition of mitochondrial oxygen consumption by AG311 was found to reduce HIF-1α stabilization by increasing oxygen tension in hypoxic conditions. Taken together, these results suggest that AG311 at least partially mediates its antitumor effect through inhibition of complex I, which could be exploited in its use as an anticancer agent.

Reengineered tricyclic anti-cancer agents.

The phenothiazine and dibenzazepine tricyclics are potent neurotropic drugs with a documented but underutilized anti-cancer side effect. Reengineering these agents (TFP, CPZ, CIP) by replacing the basic amine with a neutral polar functional group (e.g., RTC-1, RTC-2) abrogated their CNS effects as demonstrated by in vitro pharmacological assays and in vivo behavioral models. Further optimization generated several phenothiazines and dibenzazepines with improved anti-cancer potency, exemplified by RTC-5. This new lead demonstrated efficacy against a xenograft model of an EGFR driven cancer without the neurotropic effects exhibited by the parent molecules. Its effects were attributed to concomitant negative regulation of PI3K-AKT and RAS-ERK signaling.

Synthesis and biological activity of 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic agents.

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N(4)-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.

Synthesis of N(4)-(substituted phenyl)-N(4)-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells.

A series of fourteen N(4)-(substituted phenyl)-N(4)-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlorination and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nanomolar GI(50) values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6 inhibited tubulin polymerization and [(3)H]colchicine binding to tubulin. The most potent compounds were all effective in cells expressing P-glycoprotein or the ßIII isotype of tubulin, which have been associated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15 within the colchicine site.

Discovery of potent and selective inhibitors of Toxoplasma gondii thymidylate synthase for opportunistic infections.

Infection by the parasite Toxoplasma gondii (tg) can lead to toxoplasmosis in immunocompromised patients such as organ transplant, cancer and HIV/AIDS patients. The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme is crucial for nucleotide synthesis in T. gondii, and represents a potential target to combat T. gondii infection. While species selectivity with drugs has been attained for DHFR, TS is much more conserved across species and specificity is significantly more challenging. We discovered novel substituted-9H-pyrimido[4,5-b]indoles 1-3 with single-digit nanomolar Ki for tgTS, two of which, 2 and 3, are 28- and 122-fold selective over human TS (hTS). The synthesis of these compounds, and their structures in complex with tgTS-DHFR are presented along with binding measurements and cell culture data. These results show, for the very first time, that in spite of the high degree of conservation of active site residues between hTS and the parasite TS, specificity has been accomplished via novel structures and provides a new target (TS) for selective drug development against parasitic infections.

N4-(3-Bromophenyl)-7-(substituted benzyl) pyrrolo[2,3-d]pyrimidines as potent multiple receptor tyrosine kinase inhibitors: design, synthesis, and in vivo evaluation.

With the goal of developing multitargeted receptor tyrosine kinase inhibitors that display potent inhibition against PDGFRß and VEGFR-2 we designed and synthesized eleven N(4)-(3-bromophenyl)-7-(substitutedbenzyl) pyrrolo[2,3-d]pyrimidines 9a-19a. These compounds were obtained from the key intermediate N(4)-(3-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine 29. Various arylmethyl groups were regiospecifically attached at the N7 of 29 via sodium hydride induced alkylation with substituted arylmethyl halides. Compounds 11a and 19a were potent dual inhibitors of PDGFRß and VEGFR-2. In a COLO-205, in vivo tumor mouse model 11a demonstrated inhibition of tumor growth, metastasis, and tumor angiogenesis that was better than or comparable to the standard compound TSU-68 (SU6668, 8).

Diversity-oriented synthesis of a library of substituted tetrahydropyrones using oxidative carbon-hydrogen bond activation and click chemistry.

Eighteen (2RS,6RS)-2-(4-methoxyphenyl)-6-(substituted ethyl)dihydro-2H-pyran-4(3H)ones were synthesized via a DDQ-mediated oxidative carbon-hydrogen bond activation reaction. Fourteen of these tetrahydropyrans were substituted with triazoles readily assembled via azide-alkyne click-chemistry reactions. Examples of a linked benzotriazole and pyrazole motif were also prepared. To complement the structural diversity, the alcohol substrates were obtained from stereoselective reductions of the tetrahydropyrone. This library provides rapid access to structurally diverse non-natural compounds to be screened against a variety of biological targets.

Single agents with designed combination chemotherapy potential: synthesis and evaluation of substituted pyrimido[4,5-b]indoles as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents.

Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized, and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-beta (PDGFR-beta) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-beta, and hTS is better than or close to standards. In a COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.