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T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell–mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti–PD-1, to improve cancer patient outcomes.

Supplemental Figure 2. AMG 232 treatment causes p53 stabilization, and increased p21 and MDM2 proteins in p53 wild-type cells.

Supplemental Figure 3. Body weights of mice in xenograft efficacy studies

Supplemental Figure 1. AMG 232 chemical structure, and 3-D model of AMG 232 bound to MDM2 protein.

PDF file - 132KB, Figure S1. Plasma concentration-time profiles of AMG 925 in rat, dog, and cynomolgus monkey following oral administration. Figure S2. Inhibition of STAT5 and Rb phosphorylation by AMG 925 in MOLM13-Luc tumor cells in bone marrow. Table S1. Selectivity profile of AMG 925 (Kd for kinases with POC < 20 at 1 microM in KenomScan).

Supplementary Data from Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

Supplementary Figure from Targeting CD73 with AB680 (Quemliclustat), a Novel and Potent Small-Molecule CD73 Inhibitor, Restores Immune Functionality and Facilitates Antitumor Immunity

T cells play a critical role in the control of cancer. The development of immune checkpoint blockers (ICB) aimed at enhancing antitumor T-cell responses has revolutionized cancer treatment. However, durable clinical benefit is observed in only a subset of patients, prompting research efforts to focus on strategies that target multiple inhibitory signals within the tumor microenvironment (TME) to limit tumor evasion and improve patient outcomes. Adenosine has emerged as a potent immune suppressant within the TME, and CD73 is the major enzyme responsible for its extracellular production. CD73 can be co-opted within the TME to impair T-cell–mediated antitumor immunity and promote tumor growth. To target this pathway and block the formation of adenosine, we designed a novel, selective, and potent class of small-molecule inhibitors of CD73, including AB680 (quemliclustat), which is currently being tested in patients with cancer. AB680 effectively restored T-cell proliferation, cytokine secretion, and cytotoxicity that were dampened by the formation of immunosuppressive adenosine by CD73. Furthermore, in an allogeneic mixed lymphocyte reaction where CD73-derived adenosine had a dominant suppressive effect in the presence of PD-1 blockade, AB680 restored T-cell activation and function. Finally, in a preclinical mouse model of melanoma, AB680 inhibited CD73 in the TME and increased the antitumor activity of PD-1 blockade. Collectively, these data provide a rationale for the inhibition of CD73 with AB680 in combination with ICB, such as anti–PD-1, to improve cancer patient outcomes.

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 μM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

Extracellular adenosine (ADO), present in high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T cell and NK cell activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP → AMP) and CD73 (AMP → ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Extensive interrogation of structure-activity relationships (SARs), structure-based drug design, and optimization of pharmacokinetic properties culminated in the discovery of AB680, a highly potent (Ki = 5 pM), reversible, and selective inhibitor of CD73. AB680 is further characterized by very low clearance and long half-lives across preclinical species, resulting in a PK profile suitable for long-acting parenteral administration. AB680 is currently being evaluated in phase 1 clinical trials. Initial data show AB680 is well tolerated and exhibits a pharmacokinetic profile suitable for biweekly (Q2W) iv-administration in human.

CD73 is an extracellular mediator of purinergic signaling. When upregulated in the tumor microenvironment, CD73 has been implicated in the inhibition of immune function through overproduction of adenosine. Traditional efforts to inhibit CD73 have involved antibody therapy or the development of small molecules, the most potent of which mimic the acidic and ionizable structure of the enzyme's natural substrate, adenosine 5'-monophosphate (AMP). Here, we report the systematic discovery of a novel class of non-nucleotide CD73 inhibitors that are more potent than all other nonphosphonate inhibitor classes reported to date. These efforts have culminated in the discovery of 4-({5-[4-fluoro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (73, IC50 = 12 nM) and 4-({5-[4-chloro-1-(2H-indazol-6-yl)-1H-1,2,3-benzotriazol-6-yl]-1H-pyrazol-1-yl}methyl)benzonitrile (74, IC50 = 19 nM). Cocrystallization of 74 with human CD73 demonstrates a competitive binding mode. These compounds show promise for the improvement of drug-like character via the attenuation of the acidity and low membrane permeability inherent to known nucleoside inhibitors of CD73.

BackgroundTIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor expressed on natural killer (NK) cells, CD8< sup >+ T cells, CD4< sup >+ T cells and regulatory T cells (T < sub >regs). On the surface of these cells, TIGIT competes with another receptor, CD226, for shared receptor ligands (mainly CD155) that are expressed by cancer and antigen-presenting cells. Binding of CD155 to TIGIT results in immune suppression through multiple mechanisms. When TIGIT is blocked, binding of CD155 to CD226 promotes immune activation and anti-tumor immunity. We describe the preclinical characterization of AB308, a humanized wild-type IgG1 anti-TIGIT antibody that is currently undergoing clinical evaluation.MethodsBinding of AB308 to TIGIT and inhibition of the TIGIT/CD155 interaction were evaluated < i >in vitro. Functional assays were used to evaluate the immunomodulatory activity of AB308 alone or in combination with zimberelimab (anti-PD-1) or etrumadenant (a small molecule A< sub >2aA< sub >2b adenosine receptor antagonist). Surrogate Fc-silent and Fc-enabled antibodies that recognize mouse TIGIT or PD-1 were leveraged to interrogate TIGIT biology in syngeneic mouse tumor models.ResultsHuman tumor-infiltrating lymphocytes from a variety of cancer types expressed appreciable levels of TIGIT on relevant immune populations, including tumor reactive CD39< sup >+CD103< sup >+ CD8< sup >+ T cells and T< sub >regs. AB308 has a high binding affinity for human TIGIT, potently blocks the TIGIT-CD155 interaction, and induces Fcγ receptor (FcγR)-mediated signaling. In line with FcγRIII binding, AB308 also demonstrated the ability to induce NK cell-driven antibody-dependent cell-mediated cytotoxicity against TIGIT-expressing target cells. AB308 significantly increased IL-2 secretion by peripheral blood mononuclear cells activated with superantigen A, an activity that was further enhanced with zimberelimab. Blocking TIGIT with AB308 potently activated CD226 signaling in Jurkat T cells co-cultured with CD155-expressing cells, and combination of AB308 with etrumadenant in this system abrogated adenosine-mediated T cell suppression that occurred even in the presence of checkpoint inhibition. In mice, while combining Fc-silent or Fc-enabled anti-mouse TIGIT antibody with anti-PD-1 resulted in greater tumor growth inhibition than with anti-PD-1 alone, the activity of Fc-enabled anti-TIGIT was associated with intratumoral T< sub >regsdepletion.ConclusionsAB308 is a potent and highly effective anti-TIGIT antibody. Concurrent blockade of multiple immune checkpoints has the potential to confer effective and durable responses in the treatment of cancer. The data presented here support the clinical use of AB308 and provides a rationale for combination with zimberelimab and adenosine pathway blocking agents such as etrumadenant and CD73 small molecule inhibitor, AB680.Ethics ApprovalAnimal experiments were performed at Arcus Biosciences, Inc. in accordance with federal, state and Institutional guidelines and were approved by Arcus’ Institutional Animal Care and Use Committee.

CD39 (ENTPD1) is an ecto-nucleoside triphosphate diphosphohydrolase expressed widely in the tumor microenvironment (TME) responsible for catalyzing the conversion of ATP to AMP. Inhibition of CD39 enzymatic activity can promote anti-tumor immune responses by increasing the immunostimulatory substrate ATP and decreasing the formation of the product AMP, a precursor to immunoinhibitory adenosine. CD39 inhibition has been shown to have an anti-tumor effect by activating dendritic cells, macrophages, and NK cells in the TME to promote antigen presentation and inflammatory cytokine release. AB598 is a novel antibody, highly specific for human and cynomolgus CD39. AB598 potently binds to CD39 expressed on human primary myeloid cells and tumor cells and potently inhibits both soluble and membrane-bound CD39 enzymatic activity. CD39 inhibition results in increased extracellular ATP (eATP) in the TME and subsequent activation of P2X and P2Y receptors. Myeloid cells highly express both CD39 and P2X7, the P2X receptor with the weakest ligand affinity for ATP (greater than 100 μM), and most likely to be specifically activated by very high levels of eATP resulting from CD39 inhibition. Thus, the myeloid compartment has emerged as a key target associated with the therapeutic effects of CD39 inhibition. AB598 retains the ability to bind and inhibit membrane bound CD39 in the presence of high ATP (400 μM). AB598 promotes maturation of human dendritic cells in the presence of ATP, as determined by increased expression of CD83 and CD86 and decreased expression of CD14. In human macrophages treated with ATP, AB598 activates the inflammasome, resulting in the secretion of pro-inflammatory IL-18 and IL-1β. The ATP-dependent effects of AB598 support a therapeutic rationale of combining it with immunogenic therapies that induce release of ATP, such as chemotherapy and radiation, which might represent a new paradigm in the treatment of advanced solid tumors. Citation Format: Christine E. Bowman, Ada Chen, Damie Juat, Kaustubh Parashar, Julie Clor, Hema Singh, Ritu Kushwaha, Bryan Handlos, Suan Liu, Janine Kline, Xiaoning Zhao, Hyock Joo Kwon, David Green, Stephen W. Young, Ester Fernandez-Salas, Matthew J. Walters, Nigel P. Walker. Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 321.

TIGIT is an inhibitory receptor expressed primarily on NK and T cell subsets, and binding to its cognate receptor ligand, CD155, results in multiple mechanisms of immunosuppression. Blocking the TIGIT-CD155 interaction in the context of cancer promotes anti-tumor immunity. We characterized cellular subsets that TIGIT blockade may impact and the pharmacology of two anti-TIGIT antibodies - representing functional (AB308) and non-functional (AB154) Fc domain classes - undergoing clinical evaluation. Surrogate antibodies were leveraged to interrogate TIGIT biology in mouse syngeneic tumor models. Human tumor-infiltrating lymphocytes from a variety of cancer types expressed appreciable levels of TIGIT on relevant immune populations, including Tregs and CD8+ T cells with tumor reactive or pre-dysfunctional stem-like phenotypes. Both antibodies potently bound TIGIT and blocked the TIGIT-CD155 interaction as well as displayed the predicted phenotypes in terms of Fcγ receptor (FcγR) engagement. In line with FcγRIII binding, AB308 demonstrated a capacity to induce ADCC against TIGIT-expressing target cells. Combination of anti-TIGIT antibodies with other therapeutic approaches that promote T cell activation resulted in enhanced immune responses. In mice, while combining Fc-silent or Fc-enabled anti-mouse TIGIT antibody with anti-PD-1 resulted in greater tumor growth inhibition than with anti-PD-1 alone, the activity of Fc-enabled anti-TIGIT was associated with intratumoral Treg depletion. These data provide a rationale for combination with immune-activating agents and support ongoing clinical evaluation of AB154 and AB308 with biomarker strategies focused on understanding the role of Fc functionality.

Background The microenvironment of solid tumors is hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for cancer cells to survive and metastasize. The master transcriptional regulators of hypoxia-induced genes are the HIF proteins, consisting of three distinct oxygen-regulated α monomers (HIF-1α, -2α, and -3α). In normoxia, hydroxylation of HIF-2α allows for recognition by the pVHL E3-ubiquitin ligase complex and proteasomal degradation. Exposure to hypoxia, or VHL mutation or silencing, leads to HIF-2α stabilization, dimerization with HIF-1β/ARNT, and transcription of pro-tumorigenic gene sets in a variety of cancer and non-cancer cell types in the tumor microenvironment. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence suggests that inhibiting HIF-2α is an effective strategy to mitigate tumor growth, particularly in clear cell renal cell carcinoma (ccRCC), warranting further development of HIF-2α inhibitors and investigation into the role of HIF-2α in various cellular and combinatorial settings. Methods Using a suite of assays to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule inhibitors of HIF-2α. Results Optimized compounds inhibited HIF reporter transcription and VEGF secretion. Compounds that were biochemically confirmed to bind HIF-2α also inhibited HIF-2α-, but not HIF-1α-, mediated gene expression. Characterization of HIF-2α inhibition was expanded to human stromal and immune cell subsets. While compounds inhibited pro-angiogenic gene sets in endothelial cells, inhibiting HIF-2α in activated hypoxic T cells did not affect proliferation or cytokine secretion, suggesting that HIF-2α inhibitors would not impede T cell functionality in tumors. In contrast, in a M2-polarized macrophage model for suppressive tumor-associated macrophages, HIF-2α drove hypoxia-induced changes in the chemokine secretome that favored granulocytic rather than lymphocytic infiltration, an effect that was effectively reversed by HIF-2α inhibition. At the transcriptional level, mRNA-sequencing was used to define global gene sets impacted by HIF-2α inhibition in M2 macrophages. Additionally, in a set of liver, kidney, pancreatic, and colon cancer lines, CRISPR/Cas9-mediated gene editing was used to differentiate the transcriptomic profile driven by HIF-2α from that of HIF-1α or HIF-3α, allowing for the derivation of a HIF-2α-specific gene signature. Cancer cell and macrophage-derived signatures were applied to publicly available datasets to investigate cancer types, other than ccRCC, in which HIF-2α may play an important pathological role. Conclusions Collectively, these data support the development of our novel and selective HIF-2α inhibitors for the treatment of cancer and expand the indications that may benefit beyond ccRCC.

Background: The psychometric properties of the Life Orientation Test-Revised (LOT-R) have been established among college students, yet psychometric evidence is lacking for a sample of student Veterans in postsecondary education.Aims: The purpose of this study was to evaluate psychometric properties of the LOT-R for the assessment of dispositional optimism in student Veterans by using classical test theory (CTT) in conjunction with item response theory (IRT).Method: A sample of 205 student Veterans were recruited from universities across the United States. Exploratory factor analysis was conducted to test the unidimensionality of the LOT-R. A polychotomous IRT model using graded response model (GRM) was estimated. Reliability and concurrent validity of the LOT-R were tested.Results: CTT in conjunction with IRT validated that the LOT-R is a psychometrically sound unidimensional instrument for assessing the levels of dispositional optimism in student Veterans. The LOT-R was found to be associated wit...

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

We recently reported on the discovery of AMG 232, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. AMG 232 is being evaluated in human clinical trials for cancer. Continued exploration of the N-alkyl substituent of this series, in an effort to optimize interactions with the MDM2 glycine-58 shelf region, led to the discovery of sulfonamides such as compounds 31 and 38 that have similar potency, hepatocyte stability and rat pharmacokinetic properties to AMG 232.

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

The microenvironment of solid tumors is known to be hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for tumor cells to survive and metastasize. The master transcriptional regulator of hypoxia-induced genes is the Hypoxia-Inducible Factor (HIF), consisting of an oxygen-regulated alpha monomer, of which there are three isoforms (HIF-1α, HIF-2α, and HIF-3α), that can heterodimerize with a constitutively-expressed beta monomer (HIF-1β/ARNT) using Per-ARNT-SIM (PAS) protein-protein interaction domains. In normoxia, proline residues present in the oxygen-dependent degradation domain of the HIF-α subunits are hydroxylated, allowing for recognition by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex and subsequent proteasomal degradation. Upon exposure to low oxygen conditions or in the case of VHL mutation or silencing, HIF-α subunits accumulate in the cell and mediate transcription of various pro-tumorigenic gene sets. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence is mounting that suggests inhibiting HIF-2α is a valid approach to destroy tumor cells, particularly in clear cell renal carcinoma, warranting development of next-generation inhibitors. Using a suite of in vitro and in vivo assays designed to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule antagonists of HIF-2α. These compounds inhibited HIF-dependent reporter gene transcription as well as VEGF protein secretion in a human renal cell adenocarcinoma line. Compounds that were confirmed to bind the HIF-2α PAS-B domain by Microscale thermophoresis (MST) and Thermal shift assay (TSA) also significantly inhibited HIF-2α, but not HIF-1α, target gene expression in a hepatocellular carcinoma cell line (P Citation Format: Kelsey E Sivick Gauthier, Kenneth V Lawson, Dana Piovesan, Matthew J Walters, Ada Chen, Xiaoning Zhao, Cesar Meleza, Nikki Kimura, Tim Park, Steve Young, Anh Tran, Samuel L Drew, Lixia Jin, Manmohan Leleti, Elaine Ginn, Jay P Powers. A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C050. doi:10.1158/1535-7163.TARG-19-C050

Introduction: Extracellular adenosine (ADO), present at high concentrations in the tumor microenvironment (TME), suppresses immune function via inhibition of T-cell, natural killer (NK) cell, and dendritic cell (DC) activation. Intratumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates a major pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Here we present the preclinical characterization of AB680, a novel, highly potent, reversible and selective small-molecule inhibitor of CD73, currently in preclinical development as a potential antitumor agent. The link between CD73 levels present in different tissues, efficacy in mouse tumor models, plasma and tumor exposure, and projected human pharmacokinetic (PK) profile can be combined to provide an expected AB680 dosing strategy for the upcoming first-in-human clinical trial. Methods: The potency of AB680 against human CD73 was determined using CHO-CD73 cells, blood CD8+ T-cells, recombinant human CD73, and serum/plasma using either malachite green assay, AMP-Glo assay, or LCMS/MS. The selectivity of AB680 against related ecto-nucleotidases was also assessed using similar methods. Quantitation of soluble CD73 in mouse and human serum, and mouse tumor homogenates, was performed via in-house developed and validated ELISA or Western blot methods. Syngeneic mouse tumor models were established to assess the efficacy of AB680 at multiple doses. AB680 levels in plasma and tumor associated with each dosing regimen were determined via LCMS/MS. The potency of AB680 in an intratumoral setting was determined using various biochemical methods. The effects of AB680 on syngeneic tumor volumes were assessed in prophylactic and therapeutic settings. The PK properties of AB680 were evaluated in multiple preclinical species and a projected human dosing schedule for AB680 was determined via allometric scaling. Results: AB680 is a highly potent, reversible and selective inhibitor of CD73 activity (IC50 < 0.01 nM on human CD8+ T-cells), which retains its high potency in the presence of human serum. Inhibition of AMP hydrolysis by AB680 completely reversed ADO-mediated suppression of CD4+ and CD8+ T-cell effector function, as measured by cytokine secretion and proliferation. The in vivo efficacy of AB680, as measured by its ability to restrict tumor growth at doses as low as 10 mg/kg once daily, is reflective of the following parameters: 1) plasma half-life, 2) partitioning from blood to tumor compartment, 3) potency against soluble CD73 in plasma or serum, and 4) potency against membrane-bound CD73. The PK properties of AB680 in rodent and non-rodent species are characterized by very low clearance and long half-lives. The combination of in vitro potency against mouse and human soluble and membrane-bound CD73, in vivo tumor growth regulation observed in mouse models, quantification of mouse and human CD73 levels, and the projected human PK profile for AB680 have resulted in predicted human PK properties (projected half life: 4-14 days) suitable for intravenous dosing on a schedule consistent with typical monoclonal antibody dosing cycles. Conclusions: AB680 is a highly potent and selective small-molecule inhibitor of CD73 which can mitigate AMP and ADO-mediated tumor immunosuppression by potently blocking the production of ADO. AB680 exhibits a unique projected human PK profile suitable for parental administration and is expected to enter clinical development in 2018. Citation Format: Joanne B.L. Tan, Jie Chen, Elaine Ginn, Devika Ashok, Amy E Anderson, Jesus Banuelos, Kristen Zhang, Amber Pham, Timothy Park, Ada Chen, Xiaoning Zhao, Kenneth K.V. Lawson, Jenna Jeffreys, Jarek Kalisiak, Manmohan R. Leleti, Matthew J. Walters, Jay P. Powers. Preclinical pharmacokinetic and pharmacodynamic characterization of AB680, a small-molecule CD73 inhibitor for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A157.

Sentinel assays are a convenient adjunct to LC-MS purity assessment to monitor the integrity of compounds in pharmaceutical screening collections over time. To assess the stability of compounds stored both at room temperature and at -20°C in assay-ready plates that were either vacuum pack-sealed using a convenient industrial vacuum sealing method or individually sealed using conventional foil seals, a diverse collection of#126; 5,000 compounds was assayed using a robust biochemical kinase assay at intervals over a one year period. Assay results at each time point were compared to those of initial assay using a series of correlations of compound Percent of Control (POC) values as well as IC50 values of a subset of compounds in 200 nL or 500 nL plates. The fraction of hits in common between initial assays and assays at later time points ranged from 82% to 95% over one year and remained relatively constant over time with all storage temperatures or sealing methods tested. A majority of the hits that exhibited a consistent gradual trend to lower potency over one year storage were shifted to lower potency upon the rapid removal of DMSO solvent. Compound precipitation rather than compound decomposition is the likely reason for trends to lower potency for most such compounds over the storage period. Plates stored at room temperature featured a significantly higher fraction of hits that exhibited a trend to lower apparent potency than those stored at -20°C suggesting that this lower temperature is preferable for longer-term storage.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Introduction. Extracellular adenosine (ADO) suppresses immune function via inhibition of T cell and NK cell activation and is present in high concentrations in the tumor micro-environment (TME). Intra-tumoral generation of ADO depends on the sequential catabolism of ATP by two ecto-nucleotidases, CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates a major, non-redundant, pathway of ADO production in the TME and can reverse ADO-mediated immune suppression. Here we present the characterization of AB680, a novel, highly potent, reversible and selective small molecule inhibitor of CD73, currently in preclinical development as a potential anti-tumor agent. Methods. The potency of CD73 inhibitors was evaluated by measuring AMP hydrolysis by CHO-CD73 cells using a malachite green assay. Potency was also measured using human T-cells and soluble recombinant CD73. Selectivity against related ecto-nucleotidases was also assessed. In the presence of human serum, CD73 inhibition was measured by quantitation of AMP hydrolysis via luminescence. The ability of AB680 to reverse AMP-mediated immune suppression of human CD4+/CD8+ T cells was determined by adding exogenous AMP during T cell activation by anti-CD3/CD28/CD2 beads. The pharmacokinetic properties of AB680 were evaluated in multiple preclinical species. A projected human dosing schedule for AB680 was determined via allometric scaling. Results. AB680 is a highly potent, reversible and selective inhibitor of CD73 (IC50 < 0.01 nM on human CD8+ T-cells), which retains high potency in the presence of human serum. AB680 is > 10,000-fold selective against related ecto-nucleotidases and a large panel of unrelated enzymes, receptors, and ion channels. AB680 does not show significant inhibition of the major CYP450 isoforms or the hERG potassium channel. AB680 potently reverses AMP and ADO-mediated suppression of immune function in vitro. In the presence of high concentrations of AMP, AB680 robustly restored CD25 expression, IFN-γ production and proliferation of human CD4+ and CD8+ T-cells. The pharmacokinetics (PK) of AB680 were assessed in rodent and non-rodent species. The PK properties of AB680 are characterized by very low clearance and long half-lives in preclinical species, resulting in projected human PK properties suitable for intravenous (i.v.) dosing on a schedule consistent with typical mAb dosing cycles. High-dose infusions of AB680 in preclinical species were well tolerated. Conclusions. AB680 is a highly potent and selective small-molecule inhibitor of CD73 which can mitigate AMP and ADO-mediated tumor immunity by potently blocking the production of adenosine in the TME. AB680 exhibits a favorable projected human PK profile suitable for parental administration and is expected to enter clinical development in 2018. Citation Format: Kenneth V. Lawson, Lixia Jin, Jenna L. Jeffrey, Jarek Kalisiak, Fangfang Yin, Kristen Zhang, Ada Chen, Debbie Swinarski, Matt J. Walters, Steve Young, Ulrike Schindler, Powers P. Jay. Discovery and characterization of AB680, a potent and selective small-molecule CD73 inhibitor for cancer immunotherapy [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 1756.

PurposeTargeting the information security issue related to a bank loan process, this study aims to explore an approach that is user‐friendly to non‐information technology managers to be actively participative in the process of investigating the underlying information‐processing infrastructure.Design/methodology/approachA case study approach is used to analyze the safeguard gaps between the physical information assets (IAs) of a business process and their digital counterparts.FindingsThis study shows the existence of safeguard gaps between the physical IAs of a business process and their digital counterparts, explores a way of investigating such gaps, and provides (detection‐ and prevention‐oriented) managerial proposals to cope with the safeguard gap issue.Originality/valueThe existence of safeguard gaps releases (warning) signal to executives and executives should consult managers on how to manage these safeguard gaps. There are two types of managerial proposals to cope with the safeguard gap issue. The detection‐oriented proposals aim at ensuring early detection and interception of security breaches;, e.g. setting up a cross‐audit function to discover the security breaches. The prevention‐oriented proposals aim at alleviating the safeguard gaps;, e.g. re‐designing the processes of operation management and the associated risk management.

Rapid population growth and increasing water development costs have prompted many western governments to condition residential development approval on the adequacy of water supplies. We examine the effects of these regulations on housing supply in Colorado and New Mexico using fixed-effects panel regressions. Our findings suggest that price-based tools to ensure water availability may be a preferred regulatory alternative to quantity restrictions. Attempts to restrict groundwater basin access have not unambiguously corrected negative externalities related to growth. Meanwhile, Colorado cities' aggressive use of impact fees has facilitated water resource development, without limiting growth.

INTRODUCTION: In the tumor micro-environment (TME) Adenosine (ADO) dampens the immune response towards cancer cells by inhibiting the cytotoxic activity of effector cells and promoting the proliferation of immunosuppressive cells. Extracellular ADO is generated by the two ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→ADO). In immune cells ADO signals primarily through the G-protein coupled receptor A2aR. Inhibition of ADO generation and signaling have been shown to be promising therapeutic approaches for the treatment of cancer. METHODS: Potent and highly selective small molecule inhibitors for A2aR, CD73 and CD39 have been designed and synthesized by the Medicinal Chemistry Department at Arcus Biosciences. Enzymatic assays: CD39 and CD73 activity was assayed by quantitating hydrolysis of ATP or AMP, respectively. Inorganic phosphate was detected using a colorimetric (malachite green) protocol. ADO receptor assays: The potency of A2aR antagonists and selectivity towards related receptors was determined by measuring cAMP elevation in stably-expressing CHO cells following NECA (ADO mimic) stimulation. CD8+ T cell assays: Activation, proliferation and effector function of CD8+ T cells were quantified following compound treatment in the presence and absence of ATP. Mixed Lymphocyte Reaction (MLR): Cytokine levels were determined in an MLR assay after compound treatment in the presence and absence of ATP. Tumor models: CT26 and B16F10 syngeneic tumor models were used to assess the therapeutic effect of the inhibitors. RESULTS: Potent and highly selective small molecules have been generated to block either ADO generation or ADO signaling. The therapeutic potential of these molecules was assessed in CD8+ T cell assays, MLR assays and in various tumor models. CD73 inhibition blocked the conversion of AMP to ADO. CD39 inhibition blocked the conversion of ATP to AMP and A2aR inhibition abolished the increases in intracellular cAMP following ADO stimulation. The compounds are highly selective relative to related enzymes/receptors, as well as a large panel of unrelated targets. Inhibition of any one of the three targets robustly reversed adenosine-mediated inhibition of proliferation, CD25 expression, and IFN-γ and granzyme B production by human CD8+ T-cells. Robust inhibition of tumor growth in combination with anti-PD1 antibody was observed for several of these compounds. CONCLUSIONS: Highly potent and selective inhibitors of each of the 3 molecular targets involved in the ATP/adenosine pathway have been identified. Their ability to interfere with the extracellular generation of ADO and the immune-suppressive effects of ADO, as well as their effects on experimental tumor biology, have been demonstrated in various in vitro and in vivo models. Citation Format: Ulrike Schindler, Joanne B. Tan, Matt Walters, Annette Becker, Fangfang Yin, Ada Chen, Yu Chen, Wan Hsin Lim, Steve Young, Manmohan Leleti, Jay Powers, Juan C. Jaen. Small-molecule inhibitors of CD73, CD39 and A2aR: Three anti-cancer targets in the ATP/adenosine signaling pathway [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 2640. doi:10.1158/1538-7445.AM2017-2640

Introduction: The intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, depends on the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to form ADO). For this reason, a number of anti-CD73 antibodies are being advanced into clinical trials; however, to date there have been few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Ecto-nucleotidase activity was calculated using the Malachite green assay after 50-min incubation with 25µM AMP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; hCD73/CD8 T cells. Stable over-expression: hCD73/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. Human CD8 T cells enriched from buffy coats or leukopaks were pre-treated with varying concentrations of CD73 inhibitors prior to addition of 50 μM AMP + 10 μM EHNA and activated with T cell Activation/Expansion kit (Miltenyi). In some experiments, exogenous recombinant human IL12p70 (1-10 ng/mL) was added to the culture. Activation (CD25) and effector functions (Granzyme B and IFNγ) were measured by flow cytometry. CT26 cells were implanted into the shaved right flank of 7-8 week old Balb/c mice and measured three times a week starting at 7 days. Mice were enrolled into 4 cohorts and dosed according to the following conditions when tumour volume reached ~100 mm3. Group 1: 1% HPMC (sc/QD) + 2A3 (10 mg/kg; IP/Q3D) Group 2: A000830 (30 mg/kg; sc/QD) + 2A3 (10 mg/kg; IP/Q3D) Group 3: 1% HPMC (sc/QD) + RMP1-14 (10 mg/kg; IP/Q3D) Group 4: A000830 (30 mg/kg; sc/QD) + RMP1-14 (10 mg/kg; IP/Q3D) For interim analysis, single cell suspension was generated from tissues, blocked (clone 2.4G2), and stained with antibodies. For intracellular FOXP3 staining, samples were fixed and stained using FOXP3/Transcription Factor Staining Buffer Set. Non-specific blocking was performed with 20% normal rat serum prior to addition of anti-mouse FOXP3 antibody. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 and A001190 with the following IC50 values in overexpression systems: A000830: Mouse IC50 (1 nM); Human IC50 (3 nM) A001190: Mouse IC50 (n.d.); Human IC50 (0.03 nM) A000830 and A001190 also blocked AMP hydrolysis by freshly isolated human CD8 T cells at potencies comparable to the over-expression systems. In in vitro models of AMP/ADO-driven inhibition of human CD8+ T-cell activation, A000830 and A001190 showed robust rescue of CD25 expression and granzyme B production. Complete rescue of IFNγ; production was achieved by adding exogenous IL12. In vivo, A000830 was well-tolerated in mice, resulting in sustained plasma concentrations above IC90. Therapeutic dosing of A000830 to these mice in combination with an α-PD1 antibody resulted in robust CT26 tumor growth inhibition, greater than either treatment alone. Conclusions: Cumulatively, these data provide the initial characterization of a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Joanne BL Tan, Ada Chen, Manmohan Leleti, Annette Becker, Erick Lindsey, Jaroslaw Kalisiak, Jay P. Powers, Steve Young, Ulrike Schindler, Juan C. Jaen. Small-molecule inhibitors of ecto-nucleotidase CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumor models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B46.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

p53 is a critical tumor suppressor and is the most frequently inactivated gene in human cancer. Inhibition of the interaction of p53 with its negative regulator MDM2 represents a promising clinical strategy to treat p53 wild-type tumors. AMG 232 is a potential best-in-class inhibitor of the MDM2–p53 interaction and is currently in clinical trials. We characterized the activity of AMG 232 and its effect on p53 signaling in several preclinical tumor models. AMG 232 binds the MDM2 protein with picomolar affinity and robustly induces p53 activity, leading to cell-cycle arrest and inhibition of tumor cell proliferation. AMG 232 treatment inhibited the in vivo growth of several tumor xenografts and led to complete and durable regression of MDM2-amplified SJSA-1 tumors via growth arrest and induction of apoptosis. Therapeutic combination studies of AMG 232 with chemotherapies that induce DNA damage and p53 activity resulted in significantly superior antitumor efficacy and regression, and markedly increased activation of p53 signaling in tumors. These preclinical data support the further evaluation of AMG 232 in clinical trials as both a monotherapy and in combination with standard-of-care cytotoxics. Mol Cancer Ther; 14(3); 649–58. ©2015 AACR.

We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D–dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4–RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses. Mol Cancer Ther; 13(4); 880–9. ©2014 AACR.

Microsatellite instability (MSI) is caused by the dysfunction of mismatch repair genes, such as hMLH1, hMSH2. Loss of hMLH1 expression and methylation of CpG sites in hMLH1 promoter are frequently present in sporadic colorectal cancer with MSI. In this study, by transient transfection assay with constructs containing different lengths of hMLH1 promoter and a luciferase reporter gene, we located a proximal region of hMLH1 promoter, which plays a main role in regulating the gene. The fact that luciferase activities were high in all host cell lines regardless of their hMLH1 expression levels indicates that the transcription machinery is intact even in non-expressing cells. When hMLH1 promoter was in vitro methylated before transfection, the luciferase activities in the transfectants were significantly reduced. This observation indicates that methylation causes the inhibition of hMLH1 promoter activity. By electrophoretic mobility shift assay (EMSA), we identified a CCAAT box in this region, which specifically bound transcription factor CBF. Mutations in CCAAT box not only inhibited its binding to CBF factor, but also reduced its ability to drive the expression of luciferase gene. The role of CBF in activating transcription was further substantiated by inhibition of promoter activity with a plasmid expressing a dominant negative CBF-B mutant. Methylation at a CpG site two base pairs upstream of the CCAAT box inhibited the binding of CBF to CCAAT box. We conclude that methylation of an adjacent CpG site inhibits binding of the CBF transcription to the corresponding CCAAT box, and is one of the causes of hMLH1 gene silencing in colon cancer cells.

Virtual banking is broadly defined in this paper as the provision of banking services via means other than traditional physical branches. Currently, virtual banking exists in the forms of ATM, phone banking, home banking and Internet banking. Understanding people's adoption intention of virtual banking can help financial institutions to formulate appropriate marketing strategies for new forms of banking. Theory of planned behavior (TPB) and innovation diffusion were used to study the adoption intention of virtual banking in a well-developed international financial city. The study finds that the relationships were found only partially explained by the TPB. Other results are interesting and useful for the strategic planning of IT in banking.

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 μM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 μM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.

Introduction: Intra-tumoral generation of adenosine (ADO), a potent inhibitor of T-cell activation, requires the coordinated and sequential cleavage of extracellular adenosine triphosphate (ATP) by the ecto-nucleotidases CD39 (which produces adenosine monophosphate, AMP) and CD73 (which hydrolyzes AMP to ADO). For this reason, various anti-CD73 antibodies are being advanced into clinical trials; however, there are few reports of potent, selective, small-molecule CD73 inhibitors, such as those described here. Methods: Enzymatic assays: Ecto-nucleotidase activity was calculated based on the amount of inorganic phosphate (malachite green assay; absorbance at 620 nm) produced after 50-min incubation with 25μM AMP or ATP, in the presence of varying concentrations of test compound(s). The following systems were used. Endogenous expression: hCD73/SKOV-3 cells; mCD73/E771 cells. Stable over-expression: hCD73/CHO; hCD39/CHO. Transient expression: mCD73/CHO; NTPDase2/CHO; NTPDase3/CHO; NTPDase8/CHO. CD8+ T cell functional assays: Human (enriched from buffy coats or leukopaks) and mouse (isolated from spleen of C57BL/6 mice) CD8+ T cells were pretreated with CD73 inhibitor or buffer control. One hour later, cells were stimulated (anti-CD3/CD28; +/- 50 μM AMP) in the presence of ADO deaminase inhibitor (10 μM). 3 days after stimulation, cell activation (CD25), proliferation (CFSE) and effector function (IFN-γ and granzyme B) were quantified by flow cytometry. Tumor model: CT26 cells were subcutaneously implanted into Balb/c mice. When tumor volumes were ∼100 mm3, mice were enrolled into cohorts (1: vehicle control; 2: A000830; 3: PD-1 Ab; 4: A000830 + PD-1 Ab) of ≥13 mice each. Interim immune phenotyping was performed when Group 1 tumor volumes were 800-1,000 mm3. Single-cell suspensions were generated from tumors and spleens (gentleMACS). 106 cells were blocked using purified CD16/32 antibodies (clone 2.4G2) and stained with the appropriate antibody cocktails. Results: We have designed a series of potent and specific small-molecule inhibitors of human and mouse CD73, represented by A000830 (IC50 against human and mouse CD73 of 1.0 nM and 3 nM, respectively). A000830 blocked ADO generation from AMP by human ovarian cancer cells (SKOV-3) with IC50: 0.2 nM and >10,000-fold selectivity relative to other ecto-nucleotidases (CD39, NTPDase2, NTPDase3, NTPDase8) and a large panel of unrelated enzymes, receptors and ion channels. In in vitro models of ADO-driven inhibition of human and mouse CD8+ T-cell activation, A000830 showed robust rescue of proliferation, CD25 expression, and IFNγ and granzyme B production. A000830 was easily administered (and well tolerated) to mice, resulting in sustained plasma concentrations at least 1,000 times higher than its potency against mouse CD73. In the spleen of CT26 tumor-bearing Balb/c mice, CD39 expression was found mainly on natural killer (NK) cells and myeloid-lineage cells, while in the tumor CD39 was highly expressed on myeloid cells (myeloid-derived suppressor cells (MDSC) and dendritic cells (DC)) as well as CD4+ and CD8+ T cells. CD73 expression was limited (spleen and tumor) to NK cells and CD4+ and CD8+ T cells. Therapeutic dosing of A000830 to these mice (tumors ∼100 mm3 in size) in combination with anti-PD1 antibody produced robust tumor growth inhibition, greater than either treatment alone. This effect was associated with increased CD8:Treg ratios in tumors (but not spleens). Conclusions: We describe a novel class of potent and selective small-molecule CD73 inhibitors that effectively block the generation of ADO from extracellular ATP, reverse the ADO-driven inhibition of human and mouse T-cell activation, and display promising anti-tumor activity when dosed in combination with PD-1 blockade. Citation Format: Juan C. Jaen, Jay P. Powers, Ada Chen, Manmohan R. Leleti, Jarek Kalisiak, Ulrike Schindler, Joanne Bl Tan, Steve Young, Shin-Heng Chiou, Laurent Debien. Small-molecule inhibitors of CD73 promote activation of human CD8+ T cells and have profound effects on tumor growth and immune parameters in experimental tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR10.

Structure-based rational design led to the discovery of novel inhibitors of the MDM2–p53 protein–protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.

The p53 tumor suppressor is controlled by MDM2, which binds p53 and negatively regulates its transcriptional activity and stability. Many tumors overproduce MDM2 to impair p53 function. Therefore, restoration of p53 activity by inhibiting p53-MDM2 binding represents an attractive, novel approach to cancer therapy. We previously reported the discovery of AM-8553, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction (Rew et al. J. Med. Chem. 2012, 55, 4936). We report here continued optimization of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface that led to the discovery of a variety of extremely potent sulfonamides such as 14 with an IC50 of 5.3 nM in the cell proliferation assay. The compound 14 interacts specifically with the p53-binding pocket of MDM2 and releases the p53 protein from negative control. Treatment of cancer cells expressing wild-type p53 with sulfonamide 14 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis. The compound 14 showed excellent efficacy and caused tumor regression in the SJSA-1 tumor xenograft model. Citation Format: Zhihong Li, Jiasheng Fu, Yosup Rew, Michael W. Gribble, Jude Canon, Ada Chen, John Eksterowicz, Xin Huang, Lixia Jin, Mei-Chu Lo, Lawrence R. McGee, Tao Osgood, Anne Y. Saiki, Paul Shaffer, Daqing Sun, Sarah Wortman, Qiuping Ye, Dongyin Yu, Xiaoning Zhao, Jing Zhou, Jonathan D. Oliner, Steve H. Olson, Julio C. Medina. Discovery of sulfonamide-piperidinones as potent inhibitors of the MDM2-p53 protein-protein interaction. [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 3663. doi:10.1158/1538-7445.AM2015-3663

Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard care treatment, the disease eventually relapses. Activating FLT3 mutations are found in approximately 30% of AML patients. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here preclinical evaluation of AMG 925, a potent, selective and bioavailable FLT3/CDK4 dual kinase inhibitor. The compound inhibited AML xenograft tumor growth by >99% without detectable body weight loss. AMG 925 was also found to inhibit FLT3 mutants (e.g, D835Y) that are resistant to the current FLT3 inhibitors (e.g., quizartinib/AC220, sorafenib). CDK4 is a cyclinD-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-Rb pathway in cancer development has been well established. CDK4 specific inhibitors are being developed for treating Rb positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve clinical response rates. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A279. Citation Format: Kang Dai, Kathleen Keegan, Zhihong Li, Ma Ji, Cong Li, John Eksterowicz, Coberly Suzanne, David Hollenback, Margret Weidner, Justin Huard, Lingming Liang, Grace Alba, Jessica Orf, Mei-Chu Lo, Sharon Zhao, Rachel Ngo, Ada Chen, Lily Liu, Timothy Carlson, Lawrence R. McGee, Julio Medina, Alexander Kamb, Dineli Wickramasinghe. Preclinical evaluation of AMG 925, a FLT3/CDK4 dual kinase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A279.