Discovery of a novel 2-spiroproline steroid mimetic scaffold for the potent inhibition of 11ß-HSD1.

11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues, resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with a variety of ailments including metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a 3-point pharmacophore for 11ß-HSD1 that was utilized to design a 2-spiroproline derivative as a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of several leads, such as compounds 39 and 51. Importantly, deleterious hERG inhibition and pregnane X receptor induction were mitigated by the introduction of a 4-hydroxyl group to the proline ring system.

Discovery of 1'-(1-phenylcyclopropane-carbonyl)-3H-spiro[isobenzofuran-1,3'-pyrrolidin]-3-one as a novel steroid mimetic scaffold for the potent and tissue-specific inhibition of 11ß-HSD1 using a scaffold-hopping approach.

11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a three-point pharmacophore for 11ß-HSD1 that was utilized to design a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of INCB13739. Clinical evaluation of INCB13739 confirmed for the first time that tissue-specific inhibition of 11ß-HSD1 in patients with type 2 diabetes mellitus was efficacious in controlling glucose levels and reducing cardiovascular risk factors.

Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. SIGNIFICANCE: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397.

Discovery of Pemigatinib: A Potent and Selective Fibroblast Growth Factor Receptor (FGFR) Inhibitor.

Aberrant activation of FGFR has been linked to the pathogenesis of many tumor types. Selective inhibition of FGFR has emerged as a promising approach for cancer treatment. Herein, we describe the discovery of compound 38 (INCB054828, pemigatinib), a highly potent and selective inhibitor of FGFR1, FGFR2, and FGFR3 with excellent physiochemical properties and pharmacokinetic profiles. Pemigatinib has received accelerated approval from the U.S. Food and Drug Administration for the treatment of adults with previously treated, unresectable locally advanced or metastatic cholangiocarcinoma with a FGFR2 fusion or other rearrangement. Additional clinical trials are ongoing to evaluate pemigatinib in patients with FGFR alterations.

A human CD137×PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

INCB054828 (pemigatinib), a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays activity against genetically defined tumor models.

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations.

The Novel Bromodomain and Extraterminal Domain Inhibitor INCB054329 Induces Vulnerabilities in Myeloma Cells That Inform Rational Combination Strategies.

PURPOSE: Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials. EXPERIMENTAL DESIGN: We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations. RESULTS: In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK-STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone. CONCLUSIONS: Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

INCB040093 Is a Novel PI3Kδ Inhibitor for the Treatment of B Cell Lymphoid Malignancies.

Phosphatidylinositol 3-kinase delta (PI3Kδ) is a critical signaling molecule in B cells and is considered a target for development of therapies against various B cell malignancies. INCB040093 is a novel PI3Kδ small-molecule inhibitor and has demonstrated promising efficacy in patients with Hodgkin's lymphoma in clinical studies. In this study, we disclose the chemical structure and the preclinical activity of the compound. In biochemical assays, INCB040093 potently inhibits the PI3Kδ kinase, with 74- to >900-fold selectivity against other PI3K family members. In vitro and ex vivo studies using primary B cells, cell lines from B cell malignancies, and human whole blood show that INCB040093 inhibits PI3Kδ-mediated functions, including cell signaling and proliferation. INCB040093 has no significant effect on the growth of nonlymphoid cell lines and was less potent in assays that measure human T and natural killer cell proliferation and neutrophil and monocyte functions, suggesting that the impact of INCB040093 on the human immune system will likely be restricted to B cells. INCB040093 inhibits the production of macrophage-inflammatory protein-1ß (MIP-1beta) and tumor necrosis factor-ß (TNF-beta) from a B cell line, suggesting a potential effect on the tumor microenvironment. In vivo, INCB040093 demonstrates single-agent activity in inhibiting tumor growth and potentiates the antitumor growth effect of the clinically relevant chemotherapeutic agent, bendamustine, in the Pfeiffer cell xenograft model of non-Hodgkin's lymphoma. INCB040093 has a favorable exposure profile in rats and an acceptable safety margin in rats and dogs. Taken together, data presented in this report support the potential utility of orally administered INCB040093 in the treatment of B cell malignancies.

Effects of a hospital-wide intervention on emergency department crowding and quality: A prospective study.

OBJECTIVE: The objective of this study was to determine the impact of a management-supported, multimodal, hospital-wide intervention on ED crowding and quality measures. METHODS: This is a prospective descriptive study of the first 20 weeks of the intervention, with 3 years of historical controls. The study was conducted in a 600 bed adult/paediatric tertiary hospital with 80 000 ED presentations annually. ED information system data were collected on all presentations in matched 20 week periods. Multiple interventions included ED Navigator role, ED Medical Staff teaming, corporate focus with key performance indicators and dashboards, appointment of a Director of Operations, Long Length of Stay Committee and reorganisation of the flow (bed management) unit. Process outcomes were 4 h performance as a proportion of all patients and mean daily length of crowding with more than 10 inpatients awaiting beds expressed as a time. Quality outcomes were proportions of patients who did not wait and who re-presented within 72 h. RESULTS: There was a 9.1% increase in presentations and a 22.6% decrease in mean ED occupancy over the previous year. The 4 h performance improved from 56.1% (95% confidence interval [CI] 55.5-56.7) to 68.8% (95% CI 68.3-69.3) and daily crowding with more than 10 inpatients improved from 6:34 (95% CI 5:32-7:37) to 0:29 (95% CI 0:15-0:42). Did not wait improved significantly from 5.1 to 3.0% and rate of representation did not change. CONCLUSION: This prospective study shows significant improvement in ED flow without compromise in quality measures from a hospital-wide intervention requiring minimal additional resources. Further research is required on sustainability and patient outcomes beyond the ED.

Prehospital ketamine use by paramedics in the Australian Capital Territory: A 12 month retrospective analysis.

OBJECTIVE: The aim of this study was to describe prehospital use of ketamine by ACT Ambulance Service, and frequency of endotracheal intubation. METHODS: This was a retrospective study of patients receiving prehospital ketamine between 1 January and 31 December 2013. Episodes were identified from the prehospital electronic patient care records, then linkage to ED records at two receiving hospitals. Demographics, dose, indication and occasions of intubation were analysed. RESULTS: A total of 163 episodes were identified; 10 of these were excluded because of lack of identifying data or missing records (age 1-97 years [mean: 43, standard deviation: 21.7], 56% men). Median total dose was 60 mg (interquartile range 70; 5-400 mg) in three doses (interquartile range 3; 1-14 mg). For patients with a weight recorded (63%), median dose was 0.73 mg/kg. Indications were analgesia 68%, agitation/combative 25%, rapid sequence intubation 5% and others 2%. A total of 26 patients were endotracheally intubated, 11 prehospital (seven as an intended rapid sequence intubation and four combative patients with return of spontaneous circulation) and 15 in the ED. Of ED intubations, 10 were trauma patients and five were drug ingestion related. Patients receiving ketamine for combativeness were more likely to be intubated than those receiving it for analgesia (25 vs 7.2%; odds ratio: 3.46; 95% confidence interval: 1.12, 10.71). In those with a weight recorded, the mg/kg dose was not associated with subsequent intubation. CONCLUSIONS: Median dose for analgesia was comparable with other studies; dose for sedation was less than reported elsewhere. Intubation rate for patients receiving prehospital ketamine was 17%. Further study is recommended to assess the ED course of the non-intubated group of patients, and consideration should be given to non-weight-based methods of dose selection.

The Janus kinase 2 inhibitor fedratinib inhibits thiamine uptake: a putative mechanism for the onset of Wernicke's encephalopathy.

The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

Validation of standards for quantitative assessment of JAK2 c.1849G>T (p.V617F) allele burden analysis in clinical samples.

The substitution of valine with phenylalanine at amino acid 617 of the Janus kinase 2 (JAK2) gene (JAK2 p.V617F) occurs in a high proportion of patients with myeloproliferative neoplasms (MPNs). The ability to accurately measure JAK2 p.V617F allele burden is of great interest given the diagnostic relevance of the mutation and the ongoing clinical evaluation of JAK inhibitors. A main hurdle in developing quantitative assays for allele burden measurement is the unavailability of accurate standards for both assay validation and use in a standard curve for quantification. We describe our approach to the validation of standards for quantitative assessment of JAK2 p.V617F allele burden in clinical MPN samples. These standards were used in two JAK2 p.V617F assays, which were used to support clinical studies of ruxolitinib (Jakafi(®)) in myelofibrosis, a real-time polymerase chain reaction assay for initial screening of all samples, and a novel single-nucleotide polymorphism typing (SNaPshot)-based assay for samples with less than 5% mutant allele burden. Comparisons of allele burden data from clinical samples generated with these assays show a high degree of concordance with each other and with a pyrosequencing-based assay used for clinical reporting from an independent laboratory, thus providing independent validation to the accuracy of these standards.

Preclinical evaluation of local JAK1 and JAK2 inhibition in cutaneous inflammation.

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100 nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

Identification and characterization of INCB9471, an allosteric noncompetitive small-molecule antagonist of C-C chemokine receptor 5 with potent inhibitory activity against monocyte migration and HIV-1 infection.

C-C chemokine receptor 5 (CCR5) is a clinically proven target for inhibition of HIV-1 infection and a potential target for various inflammatory diseases. In this article, we describe 5-[(4-{(3S)-4-[(1R,2R)-2-ethoxy-5-(trifluoromethyl)-2,3-dihydro-1H-inden-1-yl]-3-methylpiperazin-1-yl}-4-methylpiperidin-1-yl)carbonyl]-4,6-dimethylpyrimidine dihydrochloride (INCB9471), a potent and specific inhibitor of human CCR5 that has been proven to be safe and efficacious in viral load reduction in phase I and II human clinical trails. INCB9471 was identified using a primary human monocyte-based radioligand competition binding assay. It potently inhibited macrophage inflammatory protein-1ß-induced monocyte migration and infection of peripheral blood mononuclear cells by a panel of R5-HIV-1 strains. The results from binding and signaling studies using incremental amounts of INCB9471 demonstrated INCB9471 as a noncompetitive CCR5 inhibitor. The CCR5 residues that are essential for interaction with INCB9471 were identified by site-specific mutagenesis studies. INCB9471 rapidly associates with but slowly dissociates from CCR5. When INCB9471 was compared with three CCR5 antagonists that had been tested in clinical trials, the potency of INCB9471 in blocking CCR5 ligand binding was similar to those of 4,6-dimethyl-5-{[4-methyl-4-((3S)-3-methyl-4-{(1R0-2-(methyloxy)-1-[4-(trifluoromethyl) phenyl]ethyl}-1-piperazingyl)-1-piperidinyl]carbonyl}pyrimidine (SCH-D; vicriviroc), 4-{[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxyl)methyl]-2, 5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenyl]oxy}benzoic acid hydrochloride (873140; aplaviroc), and 4,4-difluoro-N-((1S)-3-{(3-endo)-3-[3-methyl-5-(1-methylethyl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]oct-8-yl}-1-phenylpropyl)cyclohexanecarboxamide (UK427857; maraviroc). Its inhibitory activity against CCR5-mediated Ca(2+) mobilization was also similar to those of SCH-D and 873140. Further analysis suggested that INCB9471 and UK427857 may have different binding sites on CCR5. The significance of two CCR5 antagonists with different binding sites is discussed in the context of potentially overcoming drug-resistant HIV-1 strains.

Phosphorylation State-Dependent High Throughput Screening of the c-Met Kinase.

High-throughput screening (HTS) of ~50,000 chemical compounds against phosphorylated and unphosphorylated c-Met, a tyrosine kinase receptor for hepatocyte growth factor (HGF), was carried out in order to compare hit rates, hit potencies and also to explore scaffolds that might serve as potential leads targeting only the unphosphorylated form of the enzyme. The hit rate and potency for the confirmed hit molecules were higher for the unphosphoryalted form of c-Met. While the target of small molecule inhibitor discovery efforts has traditionally been the phosphorylated form, there are now examples of small molecules that target unphosphorylated kinases. Screening for inhibitors of unphosphorylated kinases may represent a complementary approach for prioritizing chemical scaffolds for hit-to-lead follow ups.

The 11-beta-hydroxysteroid dehydrogenase type 1 inhibitor INCB13739 improves hyperglycemia in patients with type 2 diabetes inadequately controlled by metformin monotherapy.

OBJECTIVE: 11-Beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) converts inactive cortisone into active cortisol, thereby amplifying intracellular glucocorticoid action. The efficacy and safety of the 11betaHSD1 inhibitor INCB13739 were assessed when added to ongoing metformin monotherapy in patients with type 2 diabetes exhibiting inadequate glycemic control (A1C 7-11%). RESEARCH DESIGN AND METHODS: This double-blind placebo-controlled paralleled study randomized 302 patients with type 2 diabetes (mean A1C 8.3%) on metformin monotherapy (mean 1.5 g/day) to receive one of five INCB13739 doses or placebo once daily for 12 weeks. The primary end point was the change in A1C at study end. Other end points included changes in fasting glucose, lipids, weight, adverse events, and safety. RESULTS: After 12 weeks, 200 mg of INCB13739 resulted in significant reductions in A1C (-0.6%), fasting plasma glucose (-24 mg/dl), and homeostasis model assessment-insulin resistance (HOMA-IR) (-24%) compared with placebo. Total cholesterol, LDL cholesterol, and triglycerides were all significantly decreased in hyperlipidemic patients. Body weight decreased relative to placebo after INCB13739 therapy. A reversible dose-dependent elevation in adrenocorticotrophic hormone, generally within the normal reference range, was observed. Basal cortisol homeostasis, testosterone in men, and free androgen index in women were unchanged by INCB13739. Adverse events were similar across all treatment groups. CONCLUSIONS: INCB13739 added to ongoing metformin therapy was efficacious and well tolerated in patients with type 2 diabetes who had inadequate glycemic control with metformin alone. 11BetaHSD1 inhibition offers a new potential approach to control glucose and cardiovascular risk factors in type 2 diabetes.

Discovery of INCB9471, a Potent, Selective, and Orally Bioavailable CCR5 Antagonist with Potent Anti-HIV-1 Activity.

To identify a CCR5 antagonist as an HIV-1 entry inhibitor, we designed a novel series of indane derivatives based on conformational considerations. Modification on the indane ring led to the discovery of compound 22a (INCB9471) that exhibited high affinity for CCR5, potent anti-HIV-1 activity, high receptor selectivity, excellent oral bioavailability, and a tolerated safety profile. INCB9471 has entered human clinical trials.

Combined inhibition of Janus kinase 1/2 for the treatment of JAK2V617F-driven neoplasms: selective effects on mutant cells and improvements in measures of disease severity.

PURPOSE: Deregulation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is a hallmark for the Philadelphia chromosome-negative myeloproliferative diseases polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We tested the efficacy of a selective JAK1/2 inhibitor in cellular and in vivo models of JAK2-driven malignancy. EXPERIMENTAL DESIGN: A novel inhibitor of JAK1/2 was characterized using kinase assays. Cellular effects of this compound were measured in cell lines bearing the JAK2V617F or JAK1V658F mutation, and its antiproliferative activity against primary polycythemiavera patient cells was determined using clonogenic assays. Antineoplastic activity in vivo was determined using a JAK2V617F-driven xenograft model, and effects of the compound on survival, organomegaly, body weight, and disease-associated inflammatory markers were measured. RESULTS: INCB16562 potently inhibited proliferation of cell lines and primary cells from PV patients carrying the JAK2V617F or JAK1V658F mutation by blocking JAK-STAT signaling and inducing apoptosis. In vivo, INCB16562 reduced malignant cell burden, reversed splenomegaly and normalized splenic architecture, improved body weight gains, and extended survival in a model of JAK2V617F-driven hematologic malignancy. Moreover, these mice suffered from markedly elevated levels of inflammatory cytokines, similar to advanced myeloproliferative disease patients, which was reversed upon treatment. CONCLUSIONS: These data showed that administration of the dual JAK1/2 inhibitor INCB16562 reduces malignant cell burden, normalizes spleen size and architecture, suppresses inflammatory cytokines, improves weight gain, and extends survival in a rodent model of JAK2V617F-driven hematologic malignancy. Thus, selective inhibitors of JAK1 and JAK2 represent a novel therapy for the patients with myeloproliferative diseases and other neoplasms associated with JAK dysregulation.

Conversion of an MMP-potent scaffold to an MMP-selective HER-2 sheddase inhibitor via scaffold hybridization and subtle P1' permutations.

A series of beta-sulfonamide piperidine hydroxamates were prepared and shown to be potent inhibitors of the human epidermal growth factor receptor-2 (HER-2) sheddase with excellent selectivity against MMP-1, -2, -3, and -9. This was achieved by exploiting subtle differences within the otherwise highly conserved S(1)(') binding pocket of the active sites within the metalloprotease family. In addition, it was discovered that the introduction of polarity to the P(1) and P(1)(') groups reduced the projected human clearance.

Isolation of polymorphic tri- and tetranucleotide microsatellite loci for the highly endangered Baw Baw frog (Philoria frosti).

We isolated 15 polymorphic microsatellite loci for the Baw Baw frog, Philoria frosti, from a genomic library enriched for (AAC)(n) and (AAAG)(n) repetitive elements. The number of alleles ranges from two to 14 per locus with the observed heterozygosity ranging from 0.04 to 0.96.