Small Molecular Weight Inhibitors Research Articles

Inhibitors of Glucosylceramide Synthase.

Glucosylceramide synthase can be targeted by high affinity small molecular weight inhibitors for the study of glycosphingolipid metabolism and function or for the treatment of glycosphingolipid storage disorders, including Gaucher and Fabry disease. This work is exemplified by the discovery and development of eliglustat tartrate, the first stand-alone small chemical entity approved for the treatment of Gaucher disease type 1. The development of inhibitors of glucosylceramide synthase that have utility for either research or clinical purposes begins with a testing funnel for screening candidate inhibitors for activity against this enzyme and for activity in lowering the content of glucosylceramide in intact cells. Two common assays for glucosylceramide synthase, one enzyme based and another cell based, are the focus of this chapter.

ADVANTAGES AND PROSPECTS OF TARGETED THERAPY IN ONCOLOGICAL PRACTICE:
 A literature review

Relevance: Cancer is the second leading cause of death globally and is expected to be responsible for approximately 19.3 million new cases
 and 10 million deaths in 2021. With an unprecedented understanding of the molecular pathways that drive the development and progression of
 human cancers, novel targeted therapies have become an exciting new development for anti-cancer medicine. These targeted therapies, also known
 as biologic therapies, have become a primary treatment modality by blocking the growth of cancer cells by specifically targeting molecules required
 for cell growth and tumorigenesis. Due to their specificity, these new therapies are expected to have better efficacy and limited adverse side effects
 compared to other treatment options, including hormonal and cytotoxic therapies.
 The study aimed to provide a detailed overview of the advantages and prospects of using targeted therapy in oncological practice.
 Мethods: The search was carried out in the following databases: Scopus, Medline, Cochrane, PubMed, ScienceDirect for 2016-2021. Sources
 were searched for the following keywords: clinical trials, immunotherapy, monoclonal antibodies, small molecular weight inhibitors, targeted therapy.
 Results: This review explores the clinical development, successes, and challenges facing targeted anti-cancer therapies, including both small
 molecule inhibitors and antibody-targeted therapies. The authors describe targeted therapies to epidermal growth factor receptor, vascular endothelial growth factor, human epidermal growth factor receptor 2, anaplastic lymphoma kinase, BRAF, T-cell mediated immune response inhibitors,
 cytotoxic T-lymphocyte-associated protein 4, and programmed cell death protein-1/PD-1 ligand.
 Conclusion: Over the past decade, there have been significant changes in cancer treatment, including targeted therapy, which has become
 more common. However, in monotherapy, targeted drugs show low activity. In addition, the selection of patients for targeted therapy remains a
 difficult task since there are not enough reliable biomarkers to predict the action of most targeted agents. This requires a deeper study of molecular
 biology, namely signaling pathways that determine the pathogenesis of oncological diseases.

Validating Cell Surface Proteases as Drug Targets for Cancer Therapy: What Do We Know, and Where Do We Go?

Simple SummaryCell surface proteases (so-called ectoproteases) are associated with cancer, and their targeting may confer valuable options for the improvement of cancer treatment outcome. Over the past 20 years, the permanent development of a multitude of inhibitors against several ectoproteases (including DPP4, FAP, APN, ADAM17, MMP2, and MMP9) has made it into clinical evaluation in haematological and solid tumours. Among them, a few show some efficacy, albeit limited, to cure cancer in the near future. This Review summarizes the efforts thus far undertaken in the development of ectoprotease inhibitors and highlights new directions for targeting ectoproteases as an additional weapon in the fight against cancer.Cell surface proteases (also known as ectoproteases) are transmembrane and membrane-bound enzymes involved in various physiological and pathological processes. Several members, most notably dipeptidyl peptidase 4 (DPP4/CD26) and its related family member fibroblast activation protein (FAP), aminopeptidase N (APN/CD13), a disintegrin and metalloprotease 17 (ADAM17/TACE), and matrix metalloproteinases (MMPs) MMP2 and MMP9, are often overexpressed in cancers and have been associated with tumour dysfunction. With multifaceted actions, these ectoproteases have been validated as therapeutic targets for cancer. Numerous inhibitors have been developed to target these enzymes, attempting to control their enzymatic activity. Even though clinical trials with these compounds did not show the expected results in most cases, the field of ectoprotease inhibitors is growing. This review summarizes the current knowledge on this subject and highlights the recent development of more effective and selective drugs targeting ectoproteases among which small molecular weight inhibitors, peptide conjugates, prodrugs, or monoclonal antibodies (mAbs) and derivatives. These promising avenues have the potential to deliver novel therapeutic strategies in the treatment of cancers.

SENP Proteases as Potential Targets for Cancer Therapy

Simple SummaryPost-translational modification—the biochemical addition of functional groups or proteins—occurs following protein biosynthesis and contributes to an increase in the functional diversity of the proteome. Post-translational modifications include SUMOylation—the covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. SUMOylation is a reversible modification, which is erased by SUMO-specific proteases (SENPs). Deregulation of SENPs leads to cellular dysfunction and is associated with various diseases, including cancer. The role of SENPs in cancer pathogenesis is expected, and thus these proteins are considered promising targets for drug design and development. In this review, we will discuss the role of SENPs, focusing on DNA repair and the cell cycle—cellular pathways malfunctioning in most cancer cells—and provide an update on advances in the development of SENP-oriented inhibitors.SUMOylation is a reversible post-translational modification (PTM) involving a covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. SUMO-specific proteases (SENPs) are cysteine proteases with isopeptidase activity facilitating the de-conjugation of SUMO proteins and thus participating in maintaining the balance between the pools of SUMOylated and unSUMOylated proteins and in SUMO recycling. Several studies have reported that SENPs’ aberrant expression is associated with the development and progression of cancer. In this review, we will discuss the role of SENPs in the pathogenesis of cancer, focusing on DNA repair and the cell cycle—cellular pathways malfunctioning in most cancer cells. The plausible role of SENPs in carcinogenesis resulted in the design and development of their inhibitors, including synthetic protein-based, peptide-based, and small molecular weight inhibitors, as well as naturally occurring compounds. Computational methods including virtual screening have been implemented to identify a number of lead structures in recent years. Some inhibitors suppressed the proliferation of prostate cancer cells in vitro and in vivo, confirming that SENPs are suitable targets for anti-cancer treatment. Further advances in the development of SENP-oriented inhibitors are anticipated toward SENP isoform-specific molecules with therapeutic potential.

Factor XII/XIIa inhibitors: Their discovery, development, and potential indications.

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its invivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.

Conversion between porcine naïve-like and primed ESCs and specific pluripotency marker identification.

Researchers currently lack standardized porcine-specific markers that would aid in distinguishing the naïve and primed states of porcine embryonic stem cells (ESCs). Here, we converted naïve-like porcine ESCs (nESCs, established in our lab) into primed-state cells, and we proposed a set of molecular criteria for evaluating the naïve porcine ESCs by comparing the two cell states. The reverse-primed porcine ESCs (rpESCs) are phenotypically stable and karyotypically intact. Alkaline phosphatase positivity and the ability to form embryonic bodies suggest that rpESCs still retain the capacity for self-renewal. Lineage-associated genes, such as Cdx2, Sox17, Eomes, Foxa, Fgf5, and Pitx2, exhibited significant expression in rpESCs. Nonetheless, LIF/3i-grown porcine ESCs treated with the small molecular weight inhibitors CHIR99021, PD0325901, and SB431542 expressed the greatest number of pluripotency marker genes, including Oct4, Sox2, Nog, Dppa5, Nr0b1, and Klf4, and at higher levels than were observed in rpESCs. Despite their general trend toward higher expression of critical pluripotency factors, the nESCs showed downregulation of Tbx3, Nanog, and c-Myc, which are considered typical naïve factors in other species. Entry of the nESCs into the developmentally primed state was also associated with a marked reduction in Lin28 expression. These findings extend the knowledge of porcine pluripotency markers and provide a backdrop for future analysis of naïve porcine pluripotency.

Toward the Discovery and Development of PSMA Targeted Inhibitors for Nuclear Medicine Applications.

Background: The rising incidence rate of prostate cancer (PCa) has promoted the development of new diagnostic and therapeutic radiopharmaceuticals during the last decades. Promising improvements have been achieved in clinical practice using prostate specific membrane antigen (PSMA) labeled agents, including specific antibodies and small molecular weight inhibitors. Focusing on molecular docking studies, this review aims to highlight the progress in the design of PSMA targeted agents for a potential use in nuclear medicine.Results: Although the first development of radiopharmaceuticals able to specifically recognize PSMA was exclusively oriented to macromolecule protein structure such as radiolabeled monoclonal antibodies and derivatives, the isolation of the crystal structure of PSMA served as the trigger for the synthesis and the further evaluation of a variety of low molecular weight inhibitors. Among the nuclear imaging probes and radiotherapeutics that have been developed and tested till today, labeled Glutamate-ureido inhibitors are the most prevalent PSMA-targeting agents for nuclear medicine applications.Conclusion: PSMA represents for researchers the most attractive target for the detection and treatment of patients affected by PCa using nuclear medicine modalities. [99mTc]MIP-1404 is considered the tracer of choice for SPECT imaging and [68Ga]PSMA-11 is the leading diagnostic for PET imaging by general consensus. [18F]DCFPyL and [18F]PSMA-1007 are clearly the emerging PET PSMA candidates for their great potential for a widespread commercial distribution. After paving the way with new imaging tools, academic and industrial R&Ds are now focusing on the development of PSMA inhibitors labeled with alpha or beta minus emitters for a theragnostic application.

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO.

NEMO is a scaffolding protein which plays an essential role in the NF-κB pathway by assembling the IKK-complex with the kinases IKKα and IKKβ. Upon activation, the IKK complex phosphorylates the IκB molecules leading to NF-κB nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-κB pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

Tannic complexes coated nanocontainers for controlled release of corrosion inhibitors in self-healing coatings

Nanocontainers with controlled release properties have been used in self-healing coatings for many years. However, the spontaneous leakage of the small molecular weight inhibitors from the nanocontainers promoted the development of nanovalves or gatekeepers to control inhibitor release. Herein, we demonstrate a facile method to encapsulate corrosion inhibitor in mesoporous silica nanoparticles (MSNs) with the help of tannic acid complexes, which endow the inhibitor loaded MSNs with pH-controlled release function. Commercial water-borne alkyd coating impregnated with 2 wt% of benzotriazole-loaded nanocontainers presented significant self-healing effect after 20 days of immersion in 0.1 M NaCl solution from both released benzotriazole and tannic acid as confirmed by electrochemical impedance spectroscopy and microscopy. The impedance modulus of coating with nanocontainers increased from 4.7 × 104 Ω cm2 to 1.8 × 105 Ω cm2 after 15 days of immersion.

Structure of Type-I Mycobacterium tuberculosis fatty acid synthase at 3.3\u2009\xc5 resolution

Tuberculosis (TB) is a devastating and rapidly spreading disease caused by Mycobacterium tuberculosis (Mtb). Therapy requires prolonged treatment with a combination of multiple agents and interruptions in the treatment regimen result in emergence and spread of multi-drug resistant (MDR) Mtb strains. MDR Mtb poses a significant global health problem, calling for urgent development of novel drugs to combat TB. Here, we report the 3.3 Å resolution structure of the ~2 MDa type-I fatty acid synthase (FAS-I) from Mtb, determined by single particle cryo-EM. Mtb FAS-I is an essential enzymatic complex that contributes to the virulence of Mtb, and thus a prime target for anti-TB drugs. The structural information for Mtb FAS-I we have obtained enables computer-based drug discovery approaches, and the resolution achieved by cryo-EM is sufficient for elucidating inhibition mechanisms by putative small molecular weight inhibitors.

Abstract SY38-03: Microenvironment dictates differential responseof primary tumor versus metastases

Abstract Metastasis is the leading cause of death in patients with advanced cancer. Activation of the metastatic cascade allows resilient cancer cells to adapt and progress in new organ microenvironments. Our work focuses on microenvironment-dependent mechanisms that control drug delivery, induce de novo resistance and promote acquired resistance to targeted therapies. By using multidisciplinary approaches with preclinical mouse models, including lymph node and brain metastasis, we gained compelling evidence revealing how stromal and cancer cell biology dictate the response to targeted therapies in the metastatic setting. Vascular and lymphatic vessels contribute to metastatic spread. We devised multiple strategies to normalize tumor vessels in order to enhance drug delivery and decrease immunosuppression (1). Preclinical and clinical studies suggest that lymph node metastases and primary tumors differentially respond to therapies. We used tumor models to address differential responses to the VEGFR2-3 inhibitors cediranib and vandetanib (2). Both compounds reduce primary tumor growth but failed to prevent progression of established lymph node metastases. Using multimodal imaging techniques, we observed that malignant cells in the lymph nodes do not promote angiogenesis but instead co-opt pre-existing vessels (3). This mechanism likely explains why anti-angiogenic therapies are ineffective in patients in the adjuvant setting. We are currently investigated novel co-option mechanisms in syngeneic models of glioblastoma. Brain tumors also display a unique vascular biology that dictates therapeutic response (4). The blood brain/tumor barrier (BBB and BTB) contribute to poor drug delivery in the central nervous system (CNS). Current therapies that control extracranial HER2+ breast cancer (BC) progression fail to inhibit brain metastasis in patients. To determine whether CNS vascular barriers are the sole cause of therapy failure, we treated mice bearing HER2+ BC brain metastasis with BKM120, a small molecular weight inhibitor of PI3K that bypasses the BBB/BTB (5). Despite similar drug distribution and target inhibition in brain and primary tumor lesions, only brain lesions progress under therapy. This phenomenon reflects de novo resistance as BC cells isolated from established brain metastases recover sensitivity to PI3K inhibition in vitro when compared to parental counterparts. In pursuit of potential resistance mechanisms in the brain tumor microenvironment, we found that 1) HER3 expression score is higher in CNS metastases when compared to extracranial metastases (including clinical samples); 2) Dual PI3K and HER3 inhibition significantly improves mouse survival when compared to monotherapies. Our data suggests that the brain tumor microenvironment unleashes multiple barriers, including poor drug delivery and de novo resistance, which prevent efficacy of targeted therapies. Collectively, our findings indicate that blocking the interactions between the tumor microenvironment and cancer cells is necessary to improve therapy regimens designed to treat and prevent metastatic progression.

Pretargeting in the context of theranostics and companion diagnostics in nuclear oncology

The concept of theranostics is well illustrated in nuclear oncology by radioactive iodide in thyroid diseases and by peptides or small molecular weight inhibitors in various cancers. It is also illustrated by radiolabeled antibodies, such as Zevalin, but radiolabeled antibodies have limitations that prompted the design of pretargeting. This review illustrates the achievements of pretargeting and focuses on its applications as theranostics or companion diagnostics. The review does not aim at exhaustivity. Examples from the already long history of pretargeting are selected to depict achievements but also outstanding issues. The idea of pretargeting, which separates antigen-binding from radionuclides, was proposed in the mid 1980s. Using avidin–biotin or bispecific antibodies and bivalent haptens, high contrast images and therapeutic efficacy were demonstrated in the clinic. However, the immunogenicity of streptavidin could not be abrogated, producing recombinant bispecific antibodies proved difficult and balancing between high tumor uptake and fast clearance of activity from normal tissues remains a challenge. These are the reasons for the continuing search for new approaches and new reagents. Some of these new approaches, particularly those based on biorthogonal chemistry or complementary oligonucleotides, are described and their potential as theranostics or companion diagnostics are discussed. The future of pretargeting remains uncertain, because clinical development is complex and cost of goods is high. However, pretargeting as a theranostic tool is clinical relevant, especially with short half-life radionuclides for PET imaging or targeted alpha-radionuclide therapy.

P-67 海洋真菌由来のHDM2-p53複合体形成阻害物質(ポスター発表の部)

P-67 海洋真菌由来のHDM2-p53複合体形成阻害物質(ポスター発表の部)

Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

Proteases and protease inhibitors were evaluated in preparations ofHeterodera glycinescysts (nHglCE) obtained from glasshouse cultures (GH) and a field (LR) population. Using a FRET-peptide library comprising 512 peptide substrate pools that detect four endoprotease types (aspartic, cysteine, metallo- and serine), we found that the relative distributions of six endoproteases within the four catalytic types were similar among GH and LR preparations. However, levels of mean protease activity ( s−1(μg nHglCE)−1) across all 512 pools varied nearly eight-fold among the preparations. This variation was not related to cyst source. These qualitative (type distribution) and quantitative relationships persisted when analysis was restricted to the top 40% (activity) pools. Analysis of the top 4% of activity pools revealed some substrate cleavage site preferences between the GH and LR proteases. GH and LR preparations also differed significantly in digestion rates of trypsin and matrix metalloprotease (MMP) substrates, with LR rates two-fold greater than GH rates for each protease. By contrast, inhibition of trypsin activity inMeloidogyne incognitaextracts by heat-denatured preparations (hHglCE) was 1.6-fold greater with GH hHglCE than with LR hHglCE. Inhibition of MMP activity was the same (>60%) for each hHglCE. Fractionation of GH and LR hHglCE preparations by RP-HPLC (CH3CN/0.1% TFA system) yielded protease inhibition profiles that were similar for GH and LR. MMP inhibitors eluted at 35-40% CH3CN, and trypsin inhibitors eluted at both 5% and 35-40% CH3CN, suggesting the possibility of peptide as well as other small molecular weight inhibitors. These discoveries illustrate the importance of examiningH. glycinescysts as a source of materials for novel nematode controls.

Activation of oncogenic pathways in classical Hodgkin lymphoma by decitabine: A rationale for combination with small molecular weight inhibitors.

DNA methylation is an epigenetic control mechanism that contributes to the specific phenotype and to the oncogenic program of virtually all tumor entities. Although efficacy of demethylating agents in classical Hodgkin lymphoma (cHL) was not specifically tested, a case of regression of relapsed metastatic cHL was described as a fortunate side‑effect of the demethylating agent 5‑azacytidine in a patient with myelodysplastic syndrome. We investigated molecular mechanisms of decitabine (5‑Aza‑dC) antitumor activity in cHL using gene expression profiling followed by gene set enrichment analysis. We found that 5‑Aza‑dC inhibits growth of cHL cell lines at clinically relevant concentrations of 0.25‑2µM. The antitumor effect of 5‑Aza‑dC was associated with induction of genes, which negatively regulate cell cycle progression (e.g. CDKN1A and GADD45A). Surprisingly, we also observed significant enrichment of pro‑survival pathways like MEK/ERK, JAK‑STAT and NF‑κB, as well as signatures comprising transcription‑activating genes. Among the upregulated pro‑survival genes were the anti‑apoptotic genes BCL2 and BCL2L1, as well as genes involved in transduction of growth and survival signals like STAT1, TLR7, CD40 and IL-6. We therefore analyzed whether interference with these pro‑survival pathways and genes would potentiate the antitumor effect of 5‑Aza‑dC. We could show that the BCL2/BCL2L1 inhibitor ABT263, the JAK‑STAT inhibitors fedratinib and SH‑4‑54, the AKT inhibitor KP372‑1, the NF‑κB inhibitor QNZ, as well as the bromodomain and extraterminal(BET) family proteins inhibitor JQ1 acted synergistically with 5‑Aza‑dC. We conclude that targeting of oncogenic pathways of cHL may improve efficacy of DNA-demethylating therapy in cHL.

MAP3K19 Is Overexpressed in COPD and Is a Central Mediator of Cigarette Smoke-Induced Pulmonary Inflammation and Lower Airway Destruction.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients.

Abstract 222: Inhibitors of mitochondrial transcription (IMT) specifically affect cancer cell proliferation

Abstract Mitochondria contain a unique DNA genome (mtDNA) that encodes a number of key components of the respiratory chain. Levels of mtDNA gene expression correlate with the metabolic requirements of mammalian cells. Recently, the role of mitochondrial function for the development and progression of cancer has been highlighted. Oncogenes have been identified to redirect mitochondrial functions to increase cancer cell survival. Increased levels of oxidative phosphorylation have also been implicated in cancer cell resistance towards drugs. In agreement with this notion, compounds that inhibit respiration may specifically target such resistant cells. Finally, it was demonstrated that inhibition of mitochondrial gene expression leads to apoptosis in rapidly dividing cells. Based on these observations, we present a completely novel principle different from known electron transfer chain blockers to treat cancer by inhibiting mtDNA transcription. To this end a study to identify and characterize inhibitors of mitochondrial transcription (IMTs) with lead-like properties was initiated. A high throughput biochemical screening campaign was performed identifying small molecular weight inhibitors of PolRMT, the mitochondrial RNA polymerase, which controls mtDNA transcription. Upon hit optimization, potent compounds with biochemical and cellular IC50s in the low nanomolar range have been identified. IMTs displayed good physicochemical and pharmacological properties. They have been shown to be inactive on other RNA polymerases proving their excellent specificity. Testing IMTs in assays for cancer cell proliferation revealed potent inhibition of sensitive cell lines (double digit nanomolar range). Interestingly, none of the tested primary non-cancer cell lines was affected by the IMTs, indicating a good therapeutic window. Testing selected IMTs in pharmacokinetic studies in vivo revealed high exposures and no detectable adverse effects (single and repeated dosing). Medicinal chemistry optimization will help to identify candidate compounds for efficacy testing in xenograft models. Citation Format: Tim Bergbrede, Axel Choidas, Raffaela Di Lucrezia, Sascha Menninger, Anke Unger, Koch Uwe, Peter Nussbaumer, Nina Bonekamp, Emily Hoberg, Victor Posse, Andrea Felser, Maria Falkenberg, Nils Göran Larsson, Claes M. Gustafsson, Bert M. Klebl. Inhibitors of mitochondrial transcription (IMT) specifically affect cancer cell proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 222.

Abstract 2823: Identification of highly selective inhibitors of cyclin-dependent kinase 12

Abstract CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors. To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models. Citation Format: Axel Choidas, Carsten Schultz-Fademrecht, Carsten Degeenhardt, Peter Habenberger, Uwe Koch, Jan E. Eickhoff, Ann Kathrin Greifenberg, Peter Nussbaumer, Matthias Geyer, Bert M. Klebl. Identification of highly selective inhibitors of cyclin-dependent kinase 12. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2823.

Phospho-Flow Cytometry Quantitation of STAT Signalling Checkpoints in Malignant and Non-Malignant Cells Identifies Patients Suitable for pSTAT3 and 5 Targeted Inhibitors

BackgroundIL-6 receptor/JAK/STAT is a major signalling pathway associated with pleiotropic functions including cell transformation and proliferation. STAT3, one of a family of 7 STAT members, sits at the apex of the signalling cascade and is a potential target for cancer therapy. Small molecular weight inhibitors either prevent STAT3 phosphorylation or target high constitutive levels of phosphorylated pSTAT3 (pSTAT3). Increased constitutive expression of pSTAT3 is a hallmark of solid tumors and is associated with increased morbidity. However recent studies suggest that this may not apply to hematological malignancies. Phospho-flow cytometry offers a novel approach for the quantitation of constitutive and cytokine induced pSTAT3 expression in subsets of hemopoietic cells. Constitutive pSTAT3 expression is not a prognostic factor for patients with either multiple myeloma (MM) or acute myeloid leukemia (AML), though we recently demonstrated that IL-6 induced pSTAT3 was associated with better prognosis in MM (χ2 =13.06; p<0.0003) (Leukemia 2015; 29:483) . There is considerable heterogeneity of pSTAT3 expression between patients which suggests that quantitation of these new biomarkers may help to identify those patients most likely to respond to STAT-targeted therapy. MethodsWe have determined constitutive and IL-6 induced pSTAT3 and 5 in malignant plasma cells of patients with MM (n=25) and for comparison, the blast cells in AML (n=8) and CD5+ B cells in chronic lymphocytic leukemia (CLL) (n=12) as well as the non-malignant B and T cells in each sample. Normal BM cells (n=9) served as a control. We have also determined pSTAT3 and 5 expression in "side population" cells (SP), the putative myeloma stem cells, and determined whether MM and AML tumor cells induce pSTAT3 on normal lymphocytes in vitro by co-culture experiments. IL-6 receptor expression was also determined on all cells.ResultsConstitutive pSTAT3 expression was increased (>5% of normal lymphocytes) in the malignant plasma cells of 44% of patients with MM but by comparison was not increased in AML or B-CLL. Constitutive pSTAT5 was increased (>5%) in 68% of MM plasma cells and 75% of AML cells and was either normal or decreased in CLL cells. Patients with increased pSTAT3 or 5 in MM cells also had high expression in autologous normal T cells. The level of constitutive pSTAT3 and 5 was not related to the disease status (relapse vs remission). In patients with MM, IL-6 induced an increased pSTAT3 expression (induced/constitutive ratio >1.5) in both MM cells and T cells in 88% of samples. IL-6 induced pSTAT3 expression in blasts of 89% of AML patients (median ratio = 79). There was no correlation between the level of pSTAT3 induced by IL-6 and IL-6 receptor expression. IL-6 did not increase pSTAT5 levels in MM, AML or CLL. SP cells in the MM cell line RPMI 8226 had lower constitutive pSTAT3 than the non-SP population and a lower IL-6 receptor expression, but had a greater response to cytokine stimulation, suggesting that SP cells may be more sensitive than non-SP cells to inhibitors of STAT phosphorylation. Addition of RPMI 8226 cells or supernatant to normal T cells caused an increase in pSTAT3, suggesting that the upregulation of pSTAT3 is at least partly due to a tumor-derived soluble agent in the microenvironment.ConclusionsThese results demonstrate the heterogeneity of constitutive and cytokine induced pSTAT3 and 5 expression in MM, AML and CLL. Elevated levels of constitutive pSTAT3 (approx 44% of MM) and pSTAT5 (88% of MM and AML) in the malignant clone were associated with high levels in autologous non-malignant T cells and are not of prognostic significance. Therefore elevated pSTAT3 and 5 may not be an exclusive hallmark of the malignant clone, as reported in solid tumors, but rather a result of the microenvironment and a high IL6-R expression. Phospho-flow cytometry enables precise quantitation of constitutive and cytokine-induced pSTAT3 and 5, and thus can identify the patients most likely to respond to direct STAT-3 or 5 inhibitor therapies. SP cells (putative MM stem cells) appear to be potentially targetable. Inhibitors targeting signalling pathways may be more efficacious if therapy is directed to patients with targetable checkpoints. DisclosuresHart:DendroCyte BioTech Pty Ltd: Equity Ownership.

Abstract C187: Cross-inhibition of Trk receptors by TAS-119, a novel Aurora A selective inhibitor, exhibits therapeutic potential in a Trk-driven cancer model

Abstract Background; Comprehensive analysis by next-generation sequencing has identified oncogenic gene rearrangement of Trk neurotrophin receptors TrkA, TrkB and TrkC (encoded by NTRK1, NTRK2, and NTRK3 gene, respectively) in multiple tumor types including lung adenocarcinoma, colorectal cancer, glioblastoma and acute myeloid leukemia. The creation of gene fusion through genomic rearrangement results in constitutive activation of the kinase domain of Trk. These recent findings encourage study of the therapeutic potential of Trk kinases as drug targets for the specific treatment of patients with oncogenic NTRK gene abnormalities. TAS-119 is a novel small molecular weight inhibitor of Aurora A kinase and currently investigated in phase 1 clinical trial as both an enhancer of microtubule stabilizing drugs and monotherapy (1-3). In addition, TAS-119 shows pan-Trk inhibition with potency similar to Aurora A inhibition. Here, we describe the in vitro and in vivo pharmacologic profile of TAS-119 as a pan-Trk inhibitor. Methods; Kinase assays were performed with purified recombinant enzymes under optimized conditions using ATP concentration at or just below the respective Km. In cell proliferation studies, cell viability was determined at 72 hour by detecting cellular ATP concentration. Analysis of protein expression including phosphorylated proteins was conducted by Western blotting. In tumor xenograft models, cells were implanted subcutaneously in nude mice. TAS-119 was administered orally with twice daily or intermittent dosing (4 days on/ 3 days off schedule) for 14 days. Results; TAS-119 inhibited TrkA activity with an IC50 of 1 nM in cell-free system, and inhibited TrkA autophosphorylation and cell proliferation of KM12C colorectal cancer cell line harbouring TPM3-TrkA fusions at sub-nM range. In the KM12C xenograft model, both continuous and intermittent treatment with TAS-119 at 60 mg/kg or 100 mg/kg b.i.d. for 14 days resulted in dose-dependent inhibition of tumor growth with good tolerability. Delays of tumor growth in xenograft model were well correlated with reduction of TrkA autophosphorylation and inhibition of downstream signal transduction like pPLCγ, pERK and pAKT within tumors. Conclusions; TAS-119 is an orally bioavailable dual-specificity kinase inhibitor developed specifically for both Trk and Aurora A inhibition with favorable profiles in animals. These preclinical data suggest that TAS-119 has therapeutic potential for cancers with Trk rearrangements. Currently, phase 1 dose-escalation clinical trial is ongoing. Although TAS-119 is primarily being developed as an inhibitor for Aurora A kinase, it should also be tested clinically in patients with tumors showing Trk gene rearrangement. 1) 24th EORTC-NCI-AACR Symposium (2012), abstract #251 and #252 2) 25th EORTC-NCI-AACR Symposium (2013), abstract #A268 and #A269 3) 26th EORTC-NCI-AACR Symposium (2014), abstract #P005 and #P178 Citation Format: Hiroshi Sootome, Akihiro Miura, Kimihiro Ito, Takamasa Suzuki, Hiroshi Hirai, Teruhiro Utsugi, Kazuhiko Yonekura. Cross-inhibition of Trk receptors by TAS-119, a novel Aurora A selective inhibitor, exhibits therapeutic potential in a Trk-driven cancer model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C187.