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Supplementary Figures S1-S6.

Purpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life.Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models.Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B, and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras.Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors. Clin Cancer Res; 17(5); 989–1000. ©2011 AACR.

Supplementary Figure 2 from Demonstration of a Genetic Therapeutic Index for Tumors Expressing Oncogenic BRAF by the Kinase Inhibitor SB-590885

Supplementary Figure 3 from Demonstration of a Genetic Therapeutic Index for Tumors Expressing Oncogenic BRAF by the Kinase Inhibitor SB-590885

Supplementary Figure 1 from Demonstration of a Genetic Therapeutic Index for Tumors Expressing Oncogenic BRAF by the Kinase Inhibitor SB-590885

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor. (Cancer Res 2006; 66(23): 11100-5)

Long-term datasets are becoming increasingly important for assessing population- and species-level responses to a changing environment. Programs that record morphological measurements of horns, antlers, and pronghorns were established in the early- to mid-20th century to collect biological information about animals that possess large horns, antlers, or pronghorns, which could be used to assess the effectiveness of conservation efforts for large mammals in North America. The general relevance of record books has been questioned because of the minimum size requirements for inclusion in a record book, which may mask trends when changes in the population occur. We compared trends in size of antlers, horns, and pronghorns through time using records from three records programs with different minimum size requirements to evaluate the influence of entry requirements on temporal trends. We also investigated whether horn, antler, or pronghorn size affected the probability of specimens being submitted to a records program. Only two of 17 categories exhibited less-pronounced trends in the record book with the highest size requirements for entry, and in two categories trends were more pronounced. Although societal interest in submitting eligible specimens increased slightly over time in one of six categories, the probability of voluntary entry was largely random and not affected by year of harvest or size of specimen. In contrast to previous criticisms, trends in record books should not be expected to represent the size of all males within a population. Instead, our evaluation indicates that the records programs we examined can provide a useful resource for assessing long-term changes in phenotypic characteristics of ungulates, but importantly, they represent the respective range of sizes within which each program collects data.

The objective of this review is to generate a synthesis of research conducted on predation of bighorn sheep (Ovis canadensis) and to suggest directions for future research relative to current knowledge gaps and a novel hypothesis. This review is primarily based on literature from the last 60 years on desert bighorn sheep (O. c. nelsoni), Rocky Mountain bighorn sheep (O. c. canadensis), and mountain lion (Puma concolor) predation. Although, many predators kill bighorn sheep, only mountain lions are currently considered to be the primary proximate cause of mortality for many bighorn sheep populations. The ultimate cause of this phenomenon has vexed wildlife managers for >40 years. There are 3 primary reasons for increased predation on bighorn sheep by mountain lions. First, there is an increased presence of mountain lions in habitats where they were historically absent or rare because of the expansion of mule deer (Odocoileus hemionus) following the extensive conversion of fire-maintained grasslands to shrublands in the late-1800s. Second, is the extirpation of the 2 dominant apex carnivores (wolves [Canis lupus] and grizzly bears [Ursus arctos]) during this same time period and a hypothesized numerical response of mountain lions to those extirpations. Finally, the response of mountain lions to the cessation of >70 years of intensive predator control has often resulted in unsustainable mountain lion-bighorn sheep ratios, especially for desert bighorn sheep. Additionally, the effect of mountain lion predation is exacerbated by declines in bighorn sheep that do not result in declines in mountain lions because of their ability to prey switch to mule deer, elk (Cervus canadensis), or domestic cattle; kleptoparasitism of mountain lions kills, by ursids and canids, resulting in higher kill rates for mountain lions; and a possible ecological trap where adaptations derived over evolutionary time are no longer adaptive because of human-induced changes in the sympatric apex predator guild. Control of mountain lions, when mountain lion-ungulate ratios are high, might be required to protect small or endangered bighorn sheep populations, and to produce bighorn sheep for restoration efforts. © 2017 The Wildlife Society.

Fitness of female ungulates is determined by neonate survival and lifetime reproductive success. Therefore, adult female ungulates should adopt behaviors and habitat selection patterns that enhance survival of neonates during parturition and lactation. Parturition site location may play an important role in neonatal mortality of desert bighorn sheep (Ovis canadensis mexicana) when lambs are especially vulnerable to predation, but parturition sites are rarely documented for this species. Our objectives were to assess environmental characteristics at desert bighorn parturition, lamb nursery, and predation sites and to assess differences in habitat characteristics between parturition sites and nursery group sites, and predation sites and nursery group sites. We used vaginal implant transmitters (VITs) to identify parturition sites and capture neonates. We then compared elevation, slope, terrain ruggedness, and visibility at parturition, nursery, and lamb predation sites with paired random sites and compared characteristics of parturition sites and lamb predation sites to those of nursery sites. When compared to random sites, odds of a site being a parturition site were highest at intermediate slopes and decreased with increasing female visibility. Odds of a site being a predation site increased with decreasing visibility. When compared to nursery group sites, odds of a site being a parturition site had a quadratic relationship with elevation and slope, with odds being highest at intermediate elevations and intermediate slopes. When we compared predation sites to nursery sites, odds of a site being a predation were highest at low elevation areas with high visibility and high elevation areas with low visibility likely because of differences in hunting strategies of coyote (Canis latrans) and puma (Puma concolor). Parturition sites were lower in elevation and slope than nursery sites. Understanding selection of parturition sites by adult females and how habitat characteristics at these sites differ from those at predation and nursery sites can provide insight into strategies employed by female desert bighorn sheep and other species during and after parturition to promote neonate survival. © 2016 The Wildlife Society.

A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.

Foraging behavior affects animal fitness and is largely dictated by the resources available to an animal. Understanding factors that affect forage resources is important for conservation and management of wildlife. Cattle sympatry is proposed to limit desert bighorn population performance, but few studies have quantified the effect of cattle foraging on bighorn forage resources or foraging behavior by desert bighorn. We estimated forage biomass for desert bighorn sheep in 2 mountain ranges: the cattle-grazed Caballo Mountains and the ungrazed San Andres Mountains, New Mexico. We recorded foraging bout efficiency of adult females by recording feeding time/step while foraging, and activity budgets of 3 age-sex classes (i.e., adult males, adult females, yearlings). We also estimated forage biomass at sites where bighorn were observed foraging. We expected lower forage biomass in the cattle-grazed Caballo range than in the ungrazed San Andres range and lower biomass at cattle-accessible versus inaccessible areas within the Caballo range. We predicted bighorn would be less efficient foragers in the Caballo range. Groundcover forage biomass was low in both ranges throughout the study (Jun 2012–Nov 2013). Browse biomass, however, was 4.7 times lower in the Caballo range versus the San Andres range. Bighorn in the Caballo range exhibited greater overall daily travel time, presumably to locate areas of higher forage abundance. By selecting areas with greater forage abundance, adult females in the Caballo range exhibited foraging bout efficiency similar to their San Andres counterparts but lower overall daily browsing time. We did not find a significant reduction in forage biomass at cattle-accessible areas in the Caballo range. Only the most rugged areas in the Caballo range had abundant forage, potentially a result of intensive historical livestock use in less rugged areas. Forage conditions in the Caballo range apparently force bighorn to increase foraging effort by feeding only in areas where adequate forage remains. © 2015 The Wildlife Society.

A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey.

A potent series of inhibitors against the B-RafV600E kinase have been developed that show excellent activity in cellular assays and good oral bioavailability in rats. The key structural features of the series are an arylsulfonamide headgroup, a thiazole core, and a fluorine ortho to the sulfonamide nitrogen.

The in vivo temporal changes of luciferase activity were investigated under the control of an hsp70 promoter in three tumour models after the application of different intensities of high-intensity focused ultrasound (HIFU). Three cell lines, SCCVII, NIH3T3 and M21 were stably transfected with a plasmid containing the hsp70 promoter and luciferase reporter gene, and tumours were subcutaneously initiated into mice. At a size of 1300 ± 234 mm3, the tumours were exposed to five intensities of continuous HIFU (802-1401-2157-3067-4133 W/cm2) for 20 sec. Bioluminescence and MR imaging were performed to assess luciferase activity and signal intensity changes in the tissue. The MRI scan protocol was pre- and post-contrast T1-wt-SE, T2-wt-FSE, DCE-MRI, diffusion-wt STEAM sequence, T2 relaxation time determination obtained on a 1.5-T GE MRI scanner. The NIH3T3 tumours showed the highest luciferase activity of 328.1 ± 7.1 fold at 24 h at a HIFU intensity of 3067 W/cm2, the M21 tumours of 3.2 ± 0.6 fold 8 hours and the SCCVII tumours 2.9 ± 0.9 fold 4 hours post-HIFU at 2157 W/cm2. The greatest increase in T2 signal intensity and T2 relaxation time of 20.7 ± 3.4% was seen in the SCCVII tumours. The highest contrast medium uptake of 10.1 ± 1.1% was noted in the M21 tumours, and 14.8 ± 1.9% in the SCCVII tumours. In all tumours, a significant increase in the diffusion coefficient was seen with increased HIFU intensity, the highest of which was 40.3 ± 4.1% in the SCCVII tumours. The three tumour cell lines stably transfected with the hsp70/luciferase gene showed differential luciferase activity, which peaked at different times after the application of HIFU and was dependant on tumour type and HIFU energy deposition.

Purpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life. Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models. Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B, and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors. Clin Cancer Res; 17(5); 989–1000. ©2011 AACR.

We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated MEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of MEK and is abrogated by the selective MEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay.

Endoscopic retrograde cholangiopancreaticography (ERCP) is the morphologic gold standard for the diagnosis of chronic pancreatitis. Magnetic Resonance Imaging (MRI) enables the visualization of not only the pancreatic duct but also the surrounding parenchyma using T2- and T1-weighted sequences before and after the application of a contrast agent. Moreover, it allows the depiction of ductal segments distal to a stenosis or occlusion. However, conventional Magnetic Resonance Cholangiopancreaticography (MRCP) was not able to achieve accuracy similar to that of ERCP. Despite many technological innovations, such as fast breath-hold acquisitions or respiratory-gated 3D sequences, this drawback could not be overcome. In recent years, secretin-enhanced MRCP has been used for the diagnosis of chronic pancreatitis. A recent study showed that secretin not only improves the visibility of the pancreatic duct and its side branches but it also enhances the diagnostic accuracy of MRCP. The sensitivity, specificity, and positive and negative predictive values were improved by the application of secretin. Moreover, the agreement between independent observers increased after the use of secretin. In addition, quantitative post-processing tools have been developed that enable the measurement of the exocrine pancreatic output non-invasively using secretin-enhanced MRCP. These tools facilitate applications, such as functional follow-up after pancreaticogastrostomy and pancreaticogastric anastomoses, evaluation of the functional status of the graft after pancreas transplantation and follow-up of pancreatic drainage procedures and duct disruption.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor. (Cancer Res 2006; 66(23): 11100-5)

Bone metastases from a vulvar carcinoma are exceptionally rare with only five reported cases in the literature. We report on a patient who was initially treated with radical vulvectomy and bilateral inguinal lymphadenectomy for a vulvar cancer (pT2, pN2 (6/37), M0; G2). Due to a positive nodal status, adjuvant radiation of the vulva and the pelvis was performed additionally. The patient presented 4 months after initial therapy with severe pain in the right humeral shaft due to a pathologic fracture based on an osteoclastic metastasis. During osteosynthetic stabilization histologic and immunohistochemical stain gave evidence of a metastasis of the known vulvar carcinoma. Bone scan showed enhancements in both humeral heads as well as the right distal femur, whereas plain radiographs confirmed further metastases in all suspected areas. In conclusion, bone metastases should be considered in the differential diagnoses of unclear osseous pain in women with a history of vulvar cancer. Immunohistochemical examinations might be important to depict the epithelial character of the tissue and allude to the metastatic nature of such rare lesions. The atypical location should alert the physician to suspect distant metastasis, rather than locoregional disease.

We studied the effects of mountain lion (Puma concolor) predation on 2 translocated populations of bighorn sheep (Ovis canadensis) in New Mexico, USA. During 1993, 32 Rocky Mountain bighorn sheep (O. c. canadensis) were translocated to Wheeler Peak Wilderness Area in northern New Mexico, and during 1992–1993, 31 desert bighorn sheep (O. c. mexicana) were translocated to Sierra Ladron in central New Mexico. We monitored both populations from release through 2000 using fixed-wing aircraft and ground and/or helicopter surveys. We determined cause of mortality for radiomarked individuals (n = 26) and calculated survival rates, cause-specific mortality rates, exponential growth rates, and lamb:ewe ratios. The post-lambing population estimates in 2000 were 180 in Wheeler Peak and 21 in Sierra Ladron. Annual adult survival was higher (P < 0.005) in the Wheeler Peak population (0.955) than in the Sierra Ladron population (0.784). Annual lamb:ewe ratios also were higher (P < 0.001) in the Wheeler Peak pop...

γ-Secretase is a multimeric complex consisted of presenilins (PSs) and three other proteins. PSs appear to be key contributors for the enzymatic center, the potential target of a number of recently developed γ-secretase inhibitors. Using radiolabeled and unlabeled inhibitors as ligands, this study was aimed to determine thein situdistribution of γ-secretase in the brain. Characterization using PS-1 knock-out mouse embryos revealed 50 and 80% reductions of γ-secretase inhibitor binding density in the heterozygous (PS-1+/–) and homozygous (PS-1–/–) embryos, respectively, relative to the wild type (PS-1+/+). The pharmacological profile from competition binding assays suggests that the ligands may target at the N- and C-terminal fragments of PS essential for γ-secretase activity. In the adult rat brain, the binding sites existed mostly in the forebrain, the cerebellum, and discrete brainstem areas and were particularly abundant in areas rich in neuronal terminals, e.g., olfactory glomeruli, CA3–hilus area, cerebellar molecular layer, and pars reticulata of the substantia nigra. In the developing rat brain, diffuse and elevated expression of binding sites occurred at the early postnatal stage relative to the adult. The possible association of binding sites with neuronal terminals in the adult brain was further investigated after olfactory deafferentation. A significant decrease with subsequent recovery of binding sites was noted in the olfactory glomeruli after chemical damage of the olfactory epithelium. The findings in this study support a physiological role of PS or γ-secretase complex in neuronal and synaptic development and plasticity.

The in vitro pharmacological profile of a novel small molecule corticotropin-releasing factor 1 (CRF1) receptor antagonist, (±) -N -[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1 H -1,2,3-triazolo[4,5- c ]pyridin-4-amine (SN003), and the characteristics of its radioligand ([3H]SN003) are described. SN003 has high affinity and selectivity for CRF1 receptors expressed in rat cortex, pituitary, and recombinant HEK293EBNA (HEK293e) cells with respective radiolabeled ovine CRF ([125I]oCRF) binding K i values of 2.5, 7.9, and 6.8 nM. SN003 was shown to be a CRF1 receptor antagonist inasmuch as it inhibited CRF-induced cAMP accumulation in human CRF1HEK293e cells and CRF-stimulated adrenocorticotropin hormone release from rat pituitary cells without agonist activities. Significant decreases in the B max of [125I]oCRF binding by SN003 suggest that this antagonist is not simply competitive. To further explore the interaction of SN003 with the CRF1receptors, [3H]SN003 binding to rat cortex and human CRF1HEK293e cell membranes was characterized and shown to be reversible and saturable, with K D values of 4.8 and 4.6 nM, and B max values of 0.142 and 7.42 pmol/mg protein, respectively. The association and dissociation rate constants of [3H]SN003 ( k +1 0.292 nM−1min−1 and k −1 0.992 × 10−2 min−1) were also assessed using human CRF1HEK293e cell membranes, giving an equilibrium dissociation constant of 3.4 nM. Moreover, [3H]SN003 binding displayed a single affinity state and insensitivity to 5′-guanylylimidodiphosphate, consistent with characteristics of antagonist binding. Incomplete inhibition of [3H]SN003 binding by CRF peptides also suggests that SN003 is not simply competitive with CRF at CRF1 receptors. The distribution of [3H]SN003 binding sites was consistent with the expression pattern of CRF1 receptors in rat brain regions. Small molecule CRF1 antagonist radioligands like [3H]SN003 should enable a better understanding of small molecule interactions with the CRF1 receptor.

One or several factors could explain sexual segregation, in which males and females of polygynous, sexually dimorphic species form separate herds during most of the year. Bighorn sheep (Ovis canadensis) are polygynous ungulates that exhibit extreme sexual dimorphism and segregate into ram and ewe herds outside of the rutting season. Four major hypotheses for sexual segregation were tested in a population of desert bighorn (O. c. mexicana) at the Red Rock Wildlife Area, New Mexico, from 1999-2001. We collected data on the size, composition, and location of ram and ewe groups during the summer period of segregation. Activity budgets were recorded for males in ram herds and females in ewe herds, and foraging selectivity was measured for males and females in mixed groups during early rut. Habitat was evaluated by measuring forage availability, ruggedness, and visibility at sites utilized by ram and ewe groups. Ram herds utilized areas with more available forage compared with ewe sites, while ewe groups preferred more rugged terrain than that used by ram groups. Ewe groups occurred much closer to free water sources than did ram groups. Bighorns in ram and ewe groups did not differ in foraging time or selectivity, nor did time spent moving, reclining, or ruminating differ between the sexes as predicted by the 'activity budget hypothesis'. The results support the predictions of the 'reproductive strategy-predation risk hypothesis', which proposes that males seek more abundant forage in order to build up body condition needed to maximize mating success (even if exposing themselves to greater predation risk), while females choose rugged terrain that minimizes predation risk to themselves and their offspring (even if sacrificing forage abundance). Female bighorns chose sites that provided access to water, also predicted by the 'reproductive strategy-predation risk hypothesis', indicating that lactation-related water requirements may constrain the movements of ewe groups and contribute to patterns of sexual segregation in desert bighorn.

Bender and Weisenberger (2005) reported that desert bighorn sheep (Ovis canadensis) on San Andres National Wildlife Refuge (SANWR), New Mexico, USA, were primarily limited by rainfall. However, they failed to mention, or were unaware, that persistent long-term predator control was used to enhance population growth at SANWR. Additionally, lamb:female ratios were collected throughout the year, rather than dates typically associated with assessing recruitment, and therefore influence of precipitation on lamb recruitment was unknown. Finally, model predictions forwarded by Bender and Weisenberger (2005), that carrying capacity of SANWR is zero when annual rainfall is

A series of 1,2,4-triazolo[1,5-a]pyrimidin-7(3H)-ones with excellent enzyme inhibition, improved isoform selectivity, and excellent inhibition of downstream phosphorylation of AKT has been identified. Several compounds in the series demonstrated potent (∼ 0.100 μM IC(50)) growth inhibition in a PTEN deficient cancer cell line.

To better understand the effects of partial cutting on arboreal lichen production within woodland caribou ( Rangifer tarandus caribou ) habitat, lichen was hand picked from 1228 branches on 307 subalpine fir ( Abies lasiocarpa ) trees in Idaho (ID), and in British Columbia (BC). Lichen biomass from partially cut stands was compared with biomass on trees from adjacent uncut stands at each site. Arboreal lichen biomass did not differ significantly between uncut and partially cut stands. Total number of branches per tree did not differ significantly between uncut and partially cut stands. Live branches had more lichen than dead branches. Species composition of arboreal lichen changed in partially cut stands compared with adjacent uncut stands. The ratio of live to dead branches was substantially different within the BC partial cut.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival". Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers. Disclosures Gao: GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients' cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients' cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.

A series of PI3K-beta selective inhibitors, imidazo[1,2-a]-pyrimidin-5(1H)-ones, has been rationally designed based on the docking model of the more potent R enantiomer of TGX-221, identified by a chiral separation, in a PI3K-beta homology model. Synthesis and SAR of this novel chemotype are described. Several compounds in the series demonstrated potent growth inhibition in a PTEN-deficient breast cancer cell line MDA-MB-468 under anchorage independent conditions.

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

Woodland caribou (Rangifer tarandus caribou) in the southern Selkirk Mountains of British Columbia shift from a diet of primarily vascular taxa during snow-free months to an arboreal lichen – conifer diet during late winter. We present evidence that caribou diets, during the early-winter transition period, are influenced by snow accumulation rates. Caribou shift to an arboreal lichen – conifer diet earlier during winters of rapid snow accumulation and forage extensively on myrtle boxwood (Pachistima myrsinites), an evergreen shrub, and other vascular plants during years of slower snow accumulation. The role of coniferous forage in early-winter food habits is examined. Forest management strategies can be developed to provide habitat that will enable caribou to forage in response to varying snow accumulation rates.

The EZH2 histone methyltransferase is frequently mutated in diffuse large B-cell lymphoma leading to increased trimethylation of histone H3 lysine 27 (H3K27me3). Drug discovery efforts have previously identified a pyridone-based chemical series of EZH2 inhibitors that potently and selectively inhibit EZH2 catalytic activity. These compounds are capable of globally decreasing H3K27me3 levels, de-repressing EZH2 target genes, and inducing growth inhibition of many lymphoma cell lines both in cell culture and in vivo. Through medicinal chemistry optimization, we have developed EZH2 inhibitors with significantly improved potency in both biochemical and cellular assays. These compounds exhibit a prolonged enzyme residence time that can be further extended in vitro through the addition of an H3K27me3 peptide. Herein, we report the biochemical and cellular activity of these new EZH2 inhibitors. Citation Format: Heidi Ott, Glenn van Aller, Jessica Ward, BaoChau Le, Cynthia Rominger, James Foley, Susan Korenchuk, Charles McHugh, Michael Butticello, Charles Blackledge, James Brackley, Joelle Burgess, Celine Duquenne, Neil Johnson, Jiri Kasparec, Louis LaFrance, Mei Li, Kenneth McNulty, Kenneth Newlander, Stuart Romeril, Stanley Schmidt, Mark Schulz, Dai-Shi Su, Dominic Suarez, Xinrong Tian, Christopher Carpenter, Juan Luengo, Ryan Kruger, Steven Knight, Michael T. McCabe. A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity. [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 5379. doi:10.1158/1538-7445.AM2015-5379

The isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are mutated in acute myelogenous leukemia, low-grade glioma, intrahepatic cholangiocarcinoma, and chondrosarcomas. IDH1 and IDH2 normally function to convert isocitrate into alpha-ketoglutarate. However, when these enzymes are mutated at select residues the mutant enzymes now convert α-KG into 2-hydroxyglutarate (2-HG). In normal cells, 2-HG levels are typically extremely low, but IDH1/2 mutant cells can accumulate up to 10 mM 2-HG. In an effort to counteract the neomorphic activity of mutant IDH enzymes, we identified and developed potent inhibitors of IDH1. The compounds inhibit IDH1 catalytic activity in biochemical assays and reduce 2-HG production in IDH1-mutant cell lines. Consistent with the fact that 2-HG inhibits α-KG dependent enzymes including histone demethylases and Tet family hydroxylases, these IDH1 inhibitors induce a decrease in several histone methylation marks and also DNA methylation. These data demonstrate that small molecule inhibitors can reverse many of the epigenetic effects of mutant IDH1. Note: This abstract was not presented at the meeting. Citation Format: Cynthia Rominger, Chad Quinn, Enoch Gao, Beth Pietrak, Alan Rendina, Angela Smallwood, Arthur Groy, Susan Korenchuk, Charles McHugh, Ken Wiggall, Alexander Reif, Stanley Schmidt, Hongwei Qi, Huizhen Zhao, Nestor Concha, Christopher Carpenter, Juan Luengo, Ryan Kruger, Benjamin Schwartz, Nicholas Adams, Michael T. McCabe. A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells. [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 3514. doi:10.1158/1538-7445.AM2015-3514

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.

We describe the binding of [125I]tyr(o)sauvagine to membranes of corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EBNA (293ECRH2 alpha) cells. The binding of [125I]tyr(o)sauvagine to CRH2 receptors was temperature, time and tissue dependent. Equilibrium was reached in 2 hr at 23 degrees C. Saturation data best fit a two-site model with affinity constants of 44 pM and 4.1 nM for high and low affinity states, respectively. The high affinity [125I]tyr(o)sauvagine binding sites were eliminated with 200 microM Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [125I]tyr(o)sauvagine binding to CRH2 alpha receptors was: urotensinsauvagine = urocortinalpha-helical CRH9-41rh-CRHo-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [125I]tyr(o)oCRH binding to CRH, receptors: urotensinurocortinr/h-CRHo-CRH = sauvaginealpha-helical CRH9-41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH1 receptor, have little or no affinity for CRH2 alpha receptors as labeled by [125I]tyr(o)sauvagine. Two selective CRH1 receptor antagonists enabled us to examine comparative densities of CRH1 and CRH2 receptors in several brain areas. We also used [125I]tyr(o)sauvagine in combination with a specific CRH1 antagonist to examine the anatomic distribution of CRH2 receptors using receptor autoradiography. With a few notable exceptions the CRH2 receptors demonstrated autoradiographically in this study match the data obtained by in situ hybridization studies on the localization of CRH2 mRNA. The anatomic overlap of the autoradiographic and in situ hybridization data suggest that CRH2 receptors are postsynaptic. This study demonstrates the utility of using [125I]tyr(o)sauvagine to study cloned CRH2 receptors expressed in cell lines. In addition, [125I]tyr(o)sauvagine used in conjunction with saturating concentrations of a specific CRH1 receptor antagonist allows the study of CRH2 receptors in brain tissues using both in vitro homogenate binding and receptor autoradiography techniques.

To compare radiographic, magnetic resonance imaging (MRI), and outpatient direct arthroscopic evaluation of cartilage in patients with osteoarthritis (OA) of the knee.Thirty-three patients with OA of the knee were evaluated by plain weight bearing radiographs and arthroscopy using a 1.9 mm arthroscope under local anesthesia. Sixteen of these patients also had MRI of the knee performed. Knee compartments were evaluated using AP weight bearing and lateral radiographs of the knee. MRI and outpatient arthroscopic grading of cartilage were performed within 2 weeks of the plain radiographs. The MRI and arthroscopic evaluations were performed independently and were graded without knowledge of the other.In patients in whom plain radiographs, MRI, and arthroscopy were compared, the plain radiographs and MRI significantly underestimated the extent of cartilage abnormalities. There was a moderate correlation between imaged cartilage scores and the arthroscopy scores (Pearson correlation coefficient = 0.40). The arthroscopic scores were reproducible with good intra and inter-observer reliability. The arthroscopic procedure was well tolerated and actually preferred over the MRI by the majority of patients. No significant complications were noted as a result of arthroscopy.Our results indicate that outpatient arthroscopic evaluation is a useful method in evaluating surface cartilage abnormalities and is more sensitive in detecting these abnormalities than either plain radiographs or MRI. Outpatient arthroscopic evaluation of cartilage appears to be a safe, sensitive, and a well tolerated tool for evaluating patients with OA of the knee and may prove to be particularly useful in evaluating response to therapeutic interventions.

Negative feedback loops are commonplace in signal transduction pathways, restoring homeostatic equilibrium. Inhibitory drugs to these pathways, by suppressing the negative feedback, can result in hyperactivation of upstream pathway components. MEK inhibitors have been reported to suppress ERK1/2 mediated activation of DUSPs and Sprouty, resulting in increased phosphorylation and activation of both Raf and Mek. This feedback response may negatively impact an inhibitor's efficacy both by increasing pathway activation and by weakening the compound binding site. Here we report on studies characterizing the effect of the MEK1/2 inhibitor GSK1120212 in preventing activation of MEK by Raf kinases, a complementary effect to its inhibition of MEK kinase activity. We conducted in vitro MEK activation assays with Raf1 or BRAF and U-MEK in the presence or absence of GSK1120212, and analyzed MEK phosphorylation by ftMSMS. We observed that GSK1120212 completely prevented Raf-dependent phosphorylation of S217 on MEK1, resulting in mono-phosphorylated (S221) MEK1 (p-MEK1). We then showed that although p-MEK1(pS221) is more active than U-MEK, it is 83-fold less active than the fully-activated diphospho-MEK1 (pp-MEK1). Our inhibition study indicated that the affinity of GSK1120212 for MEK1 was reduced by S217 phosphorylation since the IC50 for pp-MEK1 was 15.3 nM, but We then assessed whether the effect of GSK1120212 on MEK1 activating phosphorylation occurs in cells. In Sk-MEL-28, A375P, and HCT116 cells, treatment with GSK1120212 results in a transient decrease in MEK phosphorylation detected by immunoblotting; however the levels increase over time for both HCT116 and A375P. Since the phospho-MEK antibodies may not distinguish the mono from diphospho MEK, we immunoprecipitated MEK1 from lysates and analyzed MEK phosphorylation by MS. As with the in vitro reaction, GSK1120212 prevented S217 phosphorylation, but not phosphorylation of S221. As suggested by immunblotting, S221 phosphorylation increased over time, presumably reflecting the block of negative feedback mechanisms. Since GSK1120212 does not directly inhibit either RAF1 or BRAF kinase activity, we believe that GSK1120212 binds to MEK in a way that specifically blocks the accessibility of S217 to Raf kinases. This is further supported by the observation that once S217 is phosphorylated, as in pp-MEK, the affinity for GSK1120212 is reduced. S217 phosphorylation likely alters the adjacent activation loop that partially defines the compound binding site. These results suggest that in Ras and Raf mutant tumors, GSK1120212 may suppress both MEK kinase activity and partially abrogate the activating effects due to negative feedback. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 961. doi:10.1158/1538-7445.AM2011-961