Characterization Of Small Molecule Inhibitors Research Articles

Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell by inserting new copies, generating DNA damage, and triggering inflammation. Therefore, L1 inhibition could be used to treat many diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase due to the prevalence of well-characterized inhibitors of related viral enzymes. Here we present the L1 endonuclease as another target for reducing L1 activity. We characterize structurally diverse small molecule endonuclease inhibitors using computational, biochemical, and biophysical methods. We also show that these inhibitors reduce L1 retrotransposition, L1-induced DNA damage, and inflammation reinforced by L1 in senescent cells. These inhibitors could be used for further pharmacological development and as tools to better understand the life cycle of this element and its impact on disease processes.

Identification and structural characterization of small molecule inhibitors of PINK1

Mutations in PINK1 and Parkin cause early-onset Parkinson’s Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5–3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Abstract A017: Targeting Tlk2 impairs breast cancer growth and engages immune responses

Abstract The Tousled like kinases 1 and 2 (TLK1 and TLK2) are evolutionarily conserved Ser/Thr kinases that are required for genome stability and normal development in numerous organisms. Both kinases contribute to histone deposition by targeting the histone chaperones ASF1A and ASF1B and are regulated by the DNA damage response (DDR). TLK1/2 expression is ubiquitous and often elevated in human cancers with suppressed immune gene expression. In some cancer types, elevated TLK levels correlate with decreased disease-free survival. In cultured cells, TLK depletion causes replication stress, DNA damage and altered chromatin maintenance. TLK1/2 depletion increases the efficacy of DNA damage checkpoint inhibitors and PARP inhibitors and induces the Alternative-lengthening of telomeres pathway and innate immune signaling. Collectively, this data suggests that TLK activity may be a druggable anti-cancer target. To test this, we generated a conditional Tlk2 allele in mice and introduced the Mouse Mammary Tumor Virus-Polyoma Middle Tumor-antigen (MMTV-PyMT) transgene to generate breast cancers. Deletion of Tlk2 in developing breast cancer resulted in impaired primary tumor growth and a complete inhibition of lung metastasis. Characterization of the tumor microenvironment revealed elevated immune infiltration that includes activated T cells with higher levels of PD-1, which could potentially sensitize these tumors to immune checkpoint blockade. To test this possibility, we established a syngeneic breast cancer model to allow us to combine Tlk2 depletion or deletion with DDR inhibitor and anti-PD-1 therapy in vivo. Preliminary data supports the strategy of targeting Tlk2 in cancer and we will share current data from tumor models and preliminary characterization of small molecule inhibitors. Citation Format: Marina Villamor-Payá, Adrià Caballé, Jordann Smak, Mohamed-Reda Benmebarek, Camille Stephan-Otto Attolini, Travis H. Stracker. Targeting Tlk2 impairs breast cancer growth and engages immune responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A017.

Identification and biochemical characterization of small molecule inhibitors of ERK2 that target the D-recruitment site.

Extracellular signal-regulated kinase (ERK) is the culmination of a mitogen-activated protein kinase cascade that regulates cellular processes like proliferation, migration, and survival. Consequently, abnormal ERK signaling often plays a role in the tumorigenesis and metastasis of numerous cancers. ERK inhibition is a sought-after treatment for cancers, especially since clinically approved drugs that target signaling upstream of ERK often induce acquired resistance. Furthermore, the ERK2 isoform may have a differential role in various cancers from the other canonical isoform, ERK1. We demonstrate that small molecules can inhibit ERK2 catalytic and noncatalytic functions by binding to the D-recruitment site (DRS), a protein-protein interaction site distal to the enzyme active site. Using a fluorescence anisotropy-based high-throughput screening, we identify compounds that bind to the DRS and exhibit dose-dependent inhibition of ERK2 activity and ERK2 phosphorylation. We characterize the dose-dependent potency of ERK2 inhibitors using fluorescence anisotropy-based binding assays, fluorescence-based ERK2 substrate phosphorylation assays, and in vitro ERK2 activation assays. In our example, the binding of a DRS inhibitor can be prevented by mutating the DRS residue Cys-159 to serine, indicating that this residue is essential for the interaction. Resulting inhibitors from this process can be assessed in cellular and in vivo experiments for inhibition of ERK signaling and can be evaluated as potential cancer drugs.

Discovery and characterization of small molecule inhibitors of cystathionine gamma-synthase with in planta activity.

SummaryThe synthesis of essential amino acids in plants is pivotal for their viability and growth, and these cellular pathways are therefore targeted for the discovery of new molecules for weed control. Herein, we describe the discovery and design of small molecule inhibitors of cystathionine gamma‐synthase, a key enzyme in the biosynthesis of methionine. Based on in silico screening and filtering of a large molecular database followed by the in vitro selection of molecules, we identified small molecules capable of binding the target enzyme. Molecular modelling of the interaction and direct biophysical binding enabled us to explore a focussed chemical expansion set of molecules characterized by an active phenyl‐benzamide chemical group. These molecules are bio‐active and efficiently inhibit the viability of BY‐2 tobacco cells and seedlings growth of Arabidopsis thaliana on agar plates.

Explicit Treatment of Non-Michaelis-Menten and Atypical Kinetics in Early Drug Discovery*.

Biological systems are highly regulated. They are also highly resistant to sudden perturbations enabling them to maintain the dynamic equilibrium essential to sustain life. This robustness is conferred by regulatory mechanisms that influence the activity of enzymes/proteins within their cellular context to adapt to changing environmental conditions. However, the initial rules governing the study of enzyme kinetics were mostly tested and implemented for cytosolic enzyme systems that were easy to isolate and/or recombinantly express. Moreover, these enzymes lacked complex regulatory modalities. Now, with academic labs and pharmaceutical companies turning their attention to more-complex systems (for instance, multiprotein complexes, oligomeric assemblies, membrane proteins and post-translationally modified proteins), the initial axioms defined by Michaelis-Menten (MM) kinetics are rendered inadequate, and the development of a new kind of kinetic analysis to study these systems is required. This review strives to present an overview of enzyme kinetic mechanisms that are atypical and, oftentimes, do not conform to the classical MM kinetics. Further, it presents initial ideas on the design and analysis of experiments in early drug-discovery for such systems, to enable effective screening and characterisation of small-molecule inhibitors with desirable physiological outcomes.

Abstract 6403: Characterization of small molecule inhibitors of PELP1 for treating advanced breast cancer

Abstract Background: Breast cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer death in American women. BC is composed of distinct molecular subtypes, such as ER positive BC (ER+) and triple negative BC (TNBC). Development of novel effective therapies for patients with therapy resistant BC (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BC, is a prognostic indicator of poorer BC survival, and its deregulation contributes to TR-BC. The objective of this study is characterization of a small molecule inhibitor of PELP1 (SMIP34) as novel therapeutic for treating advanced BC. Methods: Yeast two-hybrid screening was used to identify PELP1 Inhibitory Peptide 1 (PIP1). Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BC proliferation confirmed using MTT assays. Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology was used to generate a library of small chemical molecules that function as peptidomimetics of PIP1. In vitro activity was assessed using CellTiter Glo, MTT, colony formation and matrigel invasion assays in multiple BC models. Mechanistic studies were conducted using Western blot, and reporter gene assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation. Results: Screening utilizing MTT assays lead to the selection of SMIP34 as an inhibitor of PELP1. SMIP34 treatment reduced cell viability at an IC50 of 3-10µM in a panel of BC cells. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Knockdown of PELP1 in BC cells significantly reduced the SMIP34 activity, confirming target specificity. Furthermore, SMIP34 treatment significantly reduced the invasiveness and colony formation of TR-BC and TNBC cells. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumor volume compared to vehicle control. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP34) displaying therapeutic efficacy against TR-BC and TNBC in vitro, ex vivo, and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin Ann Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P. Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R. Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Characterization of small molecule inhibitors of PELP1 for treating advanced breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6403.

Identification and characterization of small molecule inhibitors of the ubiquitin ligases Siah1/2 in melanoma and prostate cancer cells

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Abstract 2907: Targeting the epigenetic regulator KAT2a in cancer

Abstract Gene expression frequently requires the dynamic remodelling of the chromatin architecture to orchestrate the assembly of transcriptional protein complexes at the promoter sites of DNA. Chromatin remodelling complexes are multicomponent proteins that facilitate reorganisation of chromatin structure using the energy derived from ATP hydrolysis, and as a result play a pivotal role in controlling gene expression. Mammalian SWI/SNF complexes are examples of chromatin remodelling complexes with multiple roles in maintaining genome stability. It is estimated that 19.6% of all human tumours have mutations in SWI/SNF complexes, leading to the deregulation of gene expression as an early driver of carcinogenesis. Our interest in developing novel treatments for renal cancer focused on the role of the chromatin remodelling complex, PBAF. The BAF180 component of PBAF is mutated in 40 % of clear cell renal carcinomas and its loss is an early event in renal cell tumorigenesis. We performed an siRNA based high throughput screen to identify genes associated with genome stability that were synthetically lethal with the loss of BAF180 and identified KAT2a as a potential druggable target. KAT2a is a protein that also functions in the regulation of gene expression as a component of the SAGA histone modifying complex. It is a multifunctional protein that can both ‘read' and ‘write' acetylation marks on histone proteins through its bromodomain and a histone acetyltransferase domain respectively. We will describe our efforts to establish suitable assays to support the discovery of selective inhibitors of the bromodomain of KAT2a. The work will include the establishment of a high throughput assay to measure compound binding, crystallographic characterisation of small molecule inhibitors of the bromodomain, and our initial studies towards the target validation of the bromodomain to recapitulate the synthetic lethality observed with depletion of BAF180. Citation Format: Andrea Gunnell, Jessica Downs, Lewis Pennicott, Kay Osborn, Darren Le Grand, Katie Duffell, Hitesh Patel, Jessica Hudson, Jessica R. Booth, Simon Ward. Targeting the epigenetic regulator KAT2a in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2907.

Abstract LB-B17: Characterization of small molecule inhibitors of the Nrf2-Keap1 interaction using MicroScale Thermophoresis

Abstract The molecular complex between Keap 1 (Kelch-like ECH-associated protein) and Nrf2 (nuclear factor erythroid 2-related factor 2) plays a major role in the regulation of cyto-protective responses to oxidative stress and electrophilic agents. The Keap1-Nrf2 pathway has been established as a therapeutic target for oxidative stress-related conditions including inflammatory, cardiovascular, neurodegenerative diseases and cancer. The European Lead Factory (ELF) is a major European project generating new lead structures for drug discovery programs in the public and private sectors. This is achieved by screening molecular targets against the Joint European Compound Library (JECL) combining compounds contributed by participating pharmaceutical companies or synthesised by chemistry SMEs. Target proposals are submitted to the ELF by European academics and SMEs and selected programs are screened against the library within the European Screening Centre. Hit compounds are provided to the program owner who gains exclusive rights to exploit them for drug discovery or as novel pharmacological tools. The aim of this project was to identify non-electrophilic inhibitors of the Keap1-Nrf2 interaction. Protein-protein interaction (PPI) targets are typically difficult to drug due to large, often quite shallow interaction surfaces between proteins, which along with other factors is associated with PPI screens often returning a high proportion of false positives. Therefore, designing a high-throughput screening (HTS) triage process that confirms target engagement of hits is essential, ideally involving the use of orthogonal biophysical assays. Here we present the successful development of a label-free MicroScale Thermophoresis (MST) assay to identify inhibitors of the Keap1-Nrf2 interaction. 318,132 compounds from the JECL were tested in a fluorescence polarization assay using a fluorescein-labelled Nrf2 peptide mimic. The HTS campaign yielded a hit rate of 0.4% and many false positives compounds were deselected due to fluorescence interference or redox reactivity. Following analytical assessment, visual inspection and legal clearance, 8 compounds were selected for further characterization. Assay development indicated that label-free MST was a suitable platform to confirm target engagement prior to investing in a Chemistry program. Two structurally related hits showed a concentration-dependent binding response to the full-length Keap1 protein in MST, which was abolished after denaturation of the Keap1 protein. These compounds were competitive with one another and with an unlabeled Nrf2 peptide mimic confirming that they were able to disrupt the interaction between Keap1 and Nrf2. Both compounds contain a chiral center and synthesis of the individual enantiomers confirmed that binding was specific to the S-stereoisomer. This data package provided the confidence to initiate a full analogue program with over 110 compounds synthesized and eventually led to ligand-bound crystal structures which helped rationalize the structure-activity relationships. Citation Format: Julie M. Rainard, Angus J. Morrison, Andrew D. Pannifer, Philip S. Jones, Richard J. Mead, Stuart P. McElroy. Characterization of small molecule inhibitors of the Nrf2-Keap1 interaction using MicroScale Thermophoresis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B17.

Identification and characterization of small molecule inhibitors of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiological agent of PRRS, an economically significant disease of swine worldwide. PRRSV is poorly controlled by the currently available vaccines, and alternative control strategies are needed to help prevent the continual circulation of the virus. Previously, we developed a synthetic route for the natural compound atractylodinol and demonstrated anti-PRRSV activity in vitro. However, the synthetic route was inefficient and the yield was poor. To identify PRRSV inhibitors that could be synthesized easily and cost-effectively, we synthesized a series of atractylodinol analogs and characterized their anti-PRRSV activity in vitro. A furan-substituted bis-enyne subunit was found to be critical for PRRSV inhibition. Six analogs had potent inhibitory activity against PRRSV with 50% inhibition concentration (IC50) of 0.4–1.4 μM and 50% cytotoxic concentration (CC50) of 209–1537 μM in MARC-145 cells. Three of the most promising compounds also demonstrated significant antiviral activity and low cytotoxicity in porcine macrophages. Inhibition of PRRSV in MARC-145 cells occurred primarily at a post-entry step during PRRSV replication, between 4 and 12 h post-entry. These results suggest that atractylodinol analogs are promising antiviral candidates that could augment current PRRSV control strategies.

Abstract 196: Identification of small molecule inhibitors of the notch transcriptional activation complex

Abstract Deregulation of the Notch pathway underlies many aspects of cancer physiology depending on cell type and context. In many human cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype. Notch also plays a central role in cancer stem cells, which may underlie a role in metastasis and resistance to therapy. The important and diverse role played by Notch in cancer makes it an exceedingly attractive target for anti-neoplastic therapeutics. However, the full range of potential targets in the pathway have been under-explored. Through computer-aided drug design we explored potential ligand binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. An in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA was used for screening. We have recently reported the identification and characterization of a small molecule inhibitor of the Notch transcriptional activation complex, termed Inhibitor of Mastermind Recruitment-1 (IMR-1). Herein we report the characterization of small molecule inhibitors of Notch derived from IMR-1 that were identified through a combination of structure-activity relationship studies and molecular docking simulations. We demonstrate that these compounds inhibit the growth of Notch dependent cell lines and decrease Notch target gene transcription. Citation Format: Luisana Astudillo, Anthony Capobianco. Identification of small molecule inhibitors of the notch transcriptional activation complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2017-196

A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate N-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson's disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and S-adenosyl-l-homocysteine, revealing IC50 values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific inhibitors.

Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2–p53 interface or MdmX ((MDM4), mouse double minute 4)–p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2–MdmX really interesting new gene (RING)–RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2–MdmX RING–RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2–MdmX RING domain inhibitors)) that specifically inhibit Mdm2–MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2–MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2–MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

Abstract 5396: Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies

Abstract The three members of the Pim kinase family, Pim-1, -2, and -3, are established oncogenes and are attractive targets in hematological malignancies. We have developed multiple potent and selective scaffolds of pan-Pim inhibitors with picomolar enzymatic potency and nanomolar cellular potency. Using these agents, we have observed that the abolishment of Pim activity impairs tumor cell viability in multiple settings, both in vitro and in vivo. We have developed numerous assays to measure Pim protein levels and activity, including Pim downstream markers p-PDCD4 and p-BAD, that might be broadly applicable to human tissues. These assays have allowed us to establish a correlation between Pim protein levels and sensitivity to Pim inhibition across multiple tumor settings, in vitro. We have observed that all tested multiple myeloma (MM) cell lines express high levels of Pim-2 protein and that pan-Pim inhibition impairs viability in 90% of these lines and induces apoptosis in a subset. In acute myelogenous leukemia (AML) cell lines, sensitivity to Pim inhibition significantly correlates with Pim-1 protein expression. Numerous diffuse, large B-cell lymphoma (DLBCL) cell lines have high Pim levels and many are sensitive to Pim inhibition. We have also assessed Pim expression and activity in human tumor and normal tissues. Studies performed with myeloma cells isolated from patient bone marrow aspirates have revealed elevated Pim-2 protein levels as well as sensitivity to ex vivo dosing with Pim inhibitors, as evidenced by inhibition of PDCD4 phosphorylation. Primary patient samples from numerous other hematological tumors have also been found to have high Pim-1 or Pim-2 protein levels. To expand the possible utility of Pim inhibitors in the clinic, we have combined our molecules with numerous clinical agents, including dexamethasone, carfilzomib, and PI3K inhibitors, across multiple settings, in vitro. In all indications surveyed, we have observed that the combination of Pim molecules and these agents can lead to synergistic effects on cell viability, apoptosis and pathway signaling. In some cases, cell lines that show mild or no response to either single agent alone are sensitive to combination treatment. Collectively, our data provide a rationale for the development of Pim kinase inhibitors for use either as monotherapy or in combination with other agents in diverse tumor settings. Citation Format: Christine E. Sastri, Nadia Guerrero, Dongyin Yu, Bethany Mattson, Ken Dellamaggiore, Yajing Yang, Paul Hughes, Hui-Ling Wang, Victor Cee, Brian A. Lanman, Liping Pettus, Anthony B. Reed, Bin Wu, Ryan Wurz, Andrew Tasker, Li-Ya Huang, Daniel Branstetter, Karen Rex, Jeffrey Winston, Teresa L. Burgess, Richard Kendall, J Russell Lipford. Characterization of small molecule inhibitors of the PIM kinases in in vitro models of hematological malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5396. doi:10.1158/1538-7445.AM2015-5396

Abstract 3520: Small molecule inhibitors of ring1B-Bmi1 E3 ligase target polycomb repressive complex 1 activity and regulate cell proliferation

Abstract Emerging evidence implicates the role of Ring1B and Bmi1 in the maintenance of embryonic stem cells and regulating adult stem cells self-renewal. Both Ring1B and Bmi1 are part of Poly-comb Repressive Complex 1(PRC1) and execute gene silencing through histone H2A lysine119 ubiquitination (UbH2A K119). In addition, Bmi1 is also required for the self-renewal capacity of leukemic stem cells, suggesting that Poly-comb Group proteins may contribute to oncogenic transformation by regulating stem cell self-renewal. Targeting cancer stem cell self-renewal has been proposed as a therapeutic goal and could be an effective approach to control tumor growth. In this study, we report the development and characterization of small molecule inhibitors against Ring1B-Bmi1 complex which interfere with its E3 ligase function. We developed ligands binding to Ring1B-Bmi1 employing NMR fragment-based screening. We further performed extensive medicinal chemistry optimization and synthesized over 80 analogs of the initial fragment hit and improved its binding affinity to Ring1B-Bmi1 complex by more than 1000-fold. The activity of developed small molecule inhibitors was analyzed by cellular and biochemical studies including in vitro ubiquitination assay and quantitative analysis of p16 expression. Mechanistically, we found that our most potent compounds inhibit E3 ligase activity of Ring1B-Bmi1 at low micromolar concentrations. As expected, treatment of HeLa and leukemic cell lines with these compounds demonstrated reduced UbH2A K119 levels and increased expression of p16. In addition, presence of the lead compounds also reduces leukemic cell proliferation as determined by MTT assays. Furthermore, the comparison between knockdown effects of Ring1B/Bmi1 with small molecule inhibitors revealed on target effect of the lead compounds. To our knowledge, these compounds represent the first small molecule inhibitors that directly bind to Ring1B-Bmi1 and inhibit its E3 ubiquitin ligase activity and have a strong potential for further development into potent anticancer agents. Citation Format: Shirish Shukla, Qingjie Zhao, Weijang Ying, Felicia Gray, Kelly Vandenberg, George Lund, Bohdan Boytsov, Shihan He, Jolanta Grembecka, Tomasz Cierpicki. Small molecule inhibitors of ring1B-Bmi1 E3 ligase target polycomb repressive complex 1 activity and regulate cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2015-3520

Identification and characterisation of small molecule inhibitors of feline coronavirus replication

Feline infectious peritonitis (FIP), a feline coronavirus (FCoV) induced disease, is almost invariably fatal with median life expectancy measured in days. Current treatment options are, at best, palliative. The objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against FCoV in vitro to determine viable candidates for therapy. A resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against FCoV. Plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and CPE inhibition and IFA-based time of addition assays. Three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced CPE at low micromolar concentrations. Orthogonal assays confirmed inhibition of CPE was associated with significant reductions in viral replication. Selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were 217, 24, and 20 for chloroquine, mefloquine, and hexamethylene amiloride respectively. Preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. These results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with FIP.

Abstract 4440: Identification and characterization of small molecule inhibitors that radiosensitize K-RAS mutant rectal cancers.

Abstract Around 40% of rectal cancers harbor activating K-RAS mutations, and these mutations are associated with resistance to chemoradiotherapy. We aimed to identify small molecule inhibitors (SMIs) that synergize with ionizing radiation (IR) (“radiosensitizers”) that could be incorporated into current treatment strategies for K-RAS mutant locally advanced rectal cancer (LARC) patients. We first optimized a high-throughput assay for measuring individual and combined effects of SMIs and IR that produces similar results to the gold standard colony formation assay. Using this screening platform and K-RAS mutant rectal cancer cell lines, we tested SMIs targeting diverse signaling pathways for radiosensitizing activity and then evaluated our top hits in follow-up experiments. The two most potent radiosensitizers were the Chk1/2 inhibitor AZD7762 and the PI3K/mTOR inhibitor BEZ235. The chemotherapeutic agent 5-fluorouracil (5-FU), which is used to treat LARC, synergized with AZD7762 and enhanced radiosensitization by AZD7762 but not by BEZ235. This study is the first to compare different SMIs in combination with IR for the treatment of K-RAS mutant rectal cancer, and our findings suggest that Chk1/2 inhibitors should be evaluated in new clinical trials for LARC. Citation Format: Laura B. Kleiman, Angela Krebs, Stephen Kim, Theodore S. Hong, Kevin M. Haigis. Identification and characterization of small molecule inhibitors that radiosensitize K-RAS mutant rectal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4440. doi:10.1158/1538-7445.AM2013-4440

Abstract 3259: Novel isoquinolinone derivatives with dual PARP-1/PARP-3 inhibitory activity are highly active against pTEN mutated colorectal cancer and glioblastoma models..

Abstract Inhibitors of the poly(ADP-ribose) polymerases (PARPs) family of proteins are currently in clinical trials as potential anticancer agents. PARP-1 is a key nuclear enzyme activated by DNA strand breaks and its activation contributes to the recruitment of DNA repair and checkpoint proteins to the sites of damage. PARP-2 shows overlapping functions with PARP-1, as demonstrated by the embryonic lethality phenotype observed in double knockout mice, compared with the viability observed for single knockout of either gene. Genetic disruption in mice of PARP-2, but not of PARP-1, has been reported to affect different processes including spermatogenesis, adipogenesis, and thymocyte survival. It has been demonstrated that BRCA1 and BRCA2-deficient cell lines are sensitive towards PARP inhibition through prevention of DNA damage repair. However, recent studies have highlighted new potential genetic backgrounds in which PARP inhibition may have a therapeutic rationale. Accordingly, it has been reported that tumor cell lines bearing PTEN mutations, or with defects in mismatch repair genes are sensitive to PARP inhibitors. Here we describe the in vitro and in vivo characterization of small molecule inhibitors belonging to the isoquinolinone chemical class, with dual PARP-1 and PARP-3 inhibitory activity and which are selective versus PARP-2. Representative compounds of the class, such as NMS-P914, whose co-crystal structure with the human PARP-1 catalytic domain will be shown, and NMS-P648, are endowed with nanomolar potency against PARP-1 and PARP-3, are >100 selective versus PARP-2, possess almost complete oral bioavailability in the mouse and excellent in vitro ADMET properties across species. In cells, NMS-P914 and NMS-P648 inhibit DNA damage-induced PARP-1 activity with an IC50 of 1 nM and display single agent cytotoxicity in BRCA deficient breast tumor cell lines (IC50 < 100 nM). Interestingly, NMS-P914 and other members of the class also display similar potency against colorectal cancer and glioblastoma cell lines bearing defects in pTEN and in mismatch repair genes, and are active against glioblastoma cell lines resistant to temozolomide, irinotecan and to known PARP inhibitors such as olaparib and veliparib. In combination studies, NMS-P914 was found to be synergistic with temozolomide, both in temozolomide sensitive and resistant glioblastoma contexts. Furthermore, combination of NMS-P914 with temozolomide gives prolonged tumor regression without increasing temozolomide toxicity in vivo, showing better tolerability compared to temozolomide combination with olaparib. Citation Format: Alessia Montagnoli, Sonia Rainoldi, Stefania Mazzoleni, Antonella Ciavolella, Rosita Lupi, Elena Casale, Marina Ciomei, Enrico Pesenti, Daniele Donati, Arturo Galvani, Gianluca Papeo. Novel isoquinolinone derivatives with dual PARP-1/PARP-3 inhibitory activity are highly active against pTEN mutated colorectal cancer and glioblastoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3259. doi:10.1158/1538-7445.AM2013-3259

Identification and characterization of small molecule inhibitors of signal transducer and activator of transcription 3 (STAT3) signaling pathway by virtual screening

Inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling pathway has been considered a novel therapeutic strategy to treat human cancers that harbor aberrantly-active STAT3. In this study, a series of small molecules were identified as novel inhibitors of STAT3 signaling pathway through virtual screening. A tricyclic scaffold containing compound, 6, was identified as an inhibitor of IL-6/STAT3 signaling with an IC50 of 26.68μM. In addition, this compound inhibited Tyr705 phosphorylation of STAT3 and had no obvious effect on upstream tyrosine kinases. Thus, compound 6 is a potential lead structure and valuable for further drug development.