Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM).

Education of our future physicians is key to addressing pain and the opioid epidemic.

We explore the opioid epidemic as an example of what an educationally-driven, forward-thinking approach to healing problematic substance use might entail. We review the current state of four topics in pain and substance use disorder/opioid use disorder education and conclude each section by identifying educational initiatives that we hope may pave the way for improved clinical management of these topics in the future. Although these initiatives will be discussed explicitly in the context of undergraduate medical education, they are offered in the hope that this philosophy may be adapted for training among all healthcare disciplines. Our ultimate purpose is to detail how the education of future clinicians is essential to changing the environment that enables the problem to persist.

Assessing Aerated Vermicompost Tea Combined with Microbial Biological Control Agents for Suppression of Fusarium and Rhizoctonia.

Biological control of plant diseases is important in organic greenhouse vegetable production, where fungicide use is limited. Organic producers use microbially diverse substrates, including composts, as media for plant growth. Previous research into the impact of vermicompost on the efficacy of applied biocontrol agents is limited. An in vitro assay was developed to test the efficacy of two biological control agents in a competitive microbial background. Suppression of the pathogen Fusarium oxysporum f. sp. radicis-cucumerinum by Clonostachys rosea f. catenulata (Gliocladium catenulatum strain J1446 [Prestop]) and Bacillus subtilis strain QST 713 (Rhapsody), was assessed on agar media amended with aerated vermicompost tea (ACT). Pathogen growth was reduced more by C. rosea than ACT alone, and C. rosea was equally effective when combined with ACT. In contrast, B. subtilis reduced pathogen growth less than ACT and, when combined, reduced pathogen growth no more than ACT alone. Both biocontrol agents were similarly tested with ACT against F. oxysporum f. sp. radicis-cucumerinum and Rhizoctonia solani on cucumber and radish. Additive, neutral, and antagonistic responses, depending on host, pathogen, and biocontrol agent, were observed. ACT alone provided more consistent disease suppression on cucumber compared with B. subtilis or C. rosea. In combination, disease suppression was most often better than each biocontrol alone but not better than ACT alone. ACT had antagonistic or additive interactions with C. rosea in the radish/R. solani pathosystem, depending on the experiment. The specific and general suppression of plant diseases by biological control agents in microbially rich environments is variable and warrants further study.

PARP Inhibition Increases the Response to Chemotherapy in Uveal Melanoma.

Uveal melanoma (UM) remains without effective therapy at the metastatic stage, which is associated with BAP-1 (BRCA1 associated protein) mutations. However, no data on DNA repair capacities in UM are available. Here, we use UM patient-derived xenografts (PDXs) to study the therapeutic activity of the PARP inhibitor olaparib, alone or in combination. First, we show that the expression and the activity of PARP proteins is similar between the PDXs and the corresponding patient's tumors. In vivo experiments in the PDX models showed that olaparib was not efficient alone, but significantly increased the efficacy of dacarbazine. Finally, using reverse phase protein arrays and immunohistochemistry, we identified proteins involved in DNA repair and apoptosis as potential biomarkers predicting response to the combination of olaparib and dacarbazine. We also observed a high increase of phosphorylated YAP and TAZ proteins after dacarbazine + olaparib treatment. Our results suggest that PARP inhibition in combination with the alkylating agent dacarbazine could be of clinical interest for UM treatment. We also observe an interesting effect of dacarbazine on the Hippo pathway, confirming the importance of this pathway in UM.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment.

Dual inhibition of protein kinase C and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma.

Uveal melanoma (UM) is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified. Novel therapeutic approaches are therefore urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071. The most promising approaches were further investigated in vitro using our unique panel of UM cell lines. When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs. Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death. While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease. Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients.

Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering.

ABL1 tyrosine-kinase inhibitors (TKI) are front-line therapy for chronic myelogenous leukaemia and are among the best-known examples of targeted cancer therapeutics. However, the dynamic uptake into cells of TKIs of low molecular weight and their intracellular behaviour is unknown because of the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs (imatinib and nilotinib) inside living cells using hyperspectral stimulated Raman scattering imaging. Concentrations of both drugs were enriched over 1,000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced more than tenfold when chloroquine is used simultaneously, which suggests that chloroquine may increase the efficacy of TKIs through lysosome-mediated drug-drug interaction in addition to the commonly proposed autophagy-inhibition mechanism.

Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

SAR and biological evaluation of 3-azabicyclo[3.1.0]hexane derivatives as µ opioid ligands.

3-Azabicyclo[3.1.0]hexane compounds were designed as novel achiral µ opioid receptor ligands for the treatment of pruritus in dogs. In this paper, we describe the SAR of this class of opioid ligand, highlighting changes to the lead structure which led to compounds having picomolar binding affinity, selective for the µ receptor over δ and κ subtypes. Some subtleties of functional activity will also be described.

Neutralising antibody responses in cattle and sheep following booster vaccination with two commercial inactivated bluetongue virus serotype 8 vaccines.

Cattle and sheep that had received a primary course of vaccination with an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were booster vaccinated 6 or 12 months later with the homologous vaccine or an alternative inactivated BTV-8 vaccine and neutralising antibody responses were determined. Antibody titres to the alternative vaccine were significantly higher than to the homologous vaccine (P=0.013) in cattle. There was no significant difference between the antibody responses to alternative and homologous vaccines in sheep. These data indicate that cattle and sheep primed with one inactivated BTV-8 vaccine may be effectively boosted with an alternative commercial inactivated BTV-8 vaccine.

The antiproliferative activity of the heat shock protein 90 inhibitor IPI-504 is not dependent on NAD(P)H:quinone oxidoreductase 1 activity in vivo.

IPI-504, a water-soluble ansamycin analogue currently being investigated in clinical trials, is a potent inhibitor of the protein chaperone heat shock protein 90 (Hsp90). Inhibition of Hsp90 by IPI-504 triggers the degradation of important oncogenic client proteins. In cells, the free base of IPI-504 hydroquinone exists in a dynamic redox equilibrium with its corresponding quinone (17-AAG); the hydroquinone form binding 50 times more tightly to Hsp90. It has been proposed recently that the NAD(P)H:quinone oxidoreductase NQO1 can produce the active hydroquinone and could be essential for the activity of IPI-504. Here, we have devised a method to directly measure the intracellular ratio of hydroquinone to quinone (HQ/Q) and have applied this measurement to correlate NQO1 enzyme abundance with HQ/Q ratio and cellular activity of IPI-504 in 30 cancer cell lines. Interestingly, the intracellular HQ/Q ratio was correlated with NQO1 levels only in a subset of cell lines and overall was poorly correlated with the growth inhibitory activity of IPI-504. Although artificial overexpression of NQO1 is able to increase the level of hydroquinone and cell sensitivity to IPI-504, it has little effect on the activity of 17-amino-17-demethoxy-geldanamycin, the major active metabolite of IPI-504. This finding could provide an explanation for the biological activity of IPI-504 in xenograft models of cell lines that are not sensitive to IPI-504 in vitro. Our results suggest that NQO1 activity is not a determinant of IPI-504 activity in vivo and, therefore, unlikely to become an important resistance mechanism to IPI-504 in the clinic.

Abnormal postnatal maintenance of elevated DLK1 transcript levels in callipyge sheep.

The underlying mechanism of the callipyge muscular hypertrophy phenotype in sheep (Ovis aries) is not presently understood. This phenotype, characterized by increased glycolytic type II muscle proportion and cell size accompanied by decreased adiposity, is not visibly detectable until approximately three to eight weeks after birth. The muscular hypertrophy results from a single nucleotide change located at the telomeric end of ovine Chromosome 18, in the region between the imprinted MATERNALLY EXPRESSED GENE 3 (MEG3) and DELTA, DROSOPHILA, HOMOLOG-LIKE 1 (DLK1) genes. The callipyge phenotype is evident only when the mutation is paternally inherited by a heterozygous individual. We have examined the pre- and postnatal expression of MEG3 and DLK1 in sheep of all four possible genotypes in affected and unaffected muscles as well as in liver. Here we show that the callipyge phenotype correlates with abnormally high DLK1 expression during the postnatal period in the affected sheep and that this elevation is specific to the hypertrophy-responsive fast-twitch muscles. These results are the first to show anomalous gene expression that coincides with both the temporal and spatial distribution of the callipyge phenotype. They suggest that the effect of the callipyge mutation is to interfere with the normal postnatal downregulation of DLK1 expression.

Tissue-specific inactivation of murine M6P/IGF2R.

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and T cell- mediated immunity. M6P/IGF2R is an imprinted gene in mice with expression only from the maternal allele. Complete knockout of this gene causes neonatal lethality, thus preventing analysis of its multifunctional role postnatally. To help elucidate the biological functions of M6P/IGF2R in adulthood, we generated both complete and tissue-specific M6P/IGF2R knockout mice using the Cre/loxP system. We confirm that complete M6P/IGF2R knockout results in fetal overgrowth and neonatal lethality. In contrast, tissue-specific inactivation of this gene in either the liver or skeletal and cardiac muscle gives rise to viable animals with no obvious phenotype. The successful creation of viable tissue-specific M6P/IGF2R knockout mouse models will now allow for detailed analysis of receptor function in a number of cellular processes including brain development, carcinogenesis, lysosomal trafficking, and T cell-mediated immunity.

Identification of the single base change causing the callipyge muscle hypertrophy phenotype, the only known example of polar overdominance in mammals.

A small genetic region near the telomere of ovine chromosome 18 was previously shown to carry the mutation causing the callipyge muscle hypertrophy phenotype in sheep. Expression of this phenotype is the only known case in mammals of paternal polar overdominance gene action. A region surrounding two positional candidate genes was sequenced in animals of known genotype. Mutation detection focused on an inbred ram of callipyge phenotype postulated to have inherited chromosome segments identical-by-descent with exception of the mutated position. In support of this hypothesis, this inbred ram was homozygous over 210 Kb of sequence, except for a single heterozygous base position. This single polymorphism was genotyped in multiple families segregating the callipyge locus (CLPG), providing 100% concordance with animals of known CLPG genotype, and was unique to descendants of the founder animal. The mutation lies in a region of high homology among mouse, sheep, cattle, and humans, but not in any previously identified expressed transcript. A substantial open reading frame exists in the sheep sequence surrounding the mutation, although this frame is not conserved among species. Initial functional analysis indicates sequence encompassing the mutation is part of a novel transcript expressed in sheep fetal muscle we have named CLPG1.

Enhanced Electronic Conjugation in Anthracene-Linked Porphyrins.

Despite their larger separation, the porphyrin groups in 1 are more strongly conjugated than in analogous compounds in which butadiyne is the bridging unit. This stronger electronic coupling in 1 results in a bathochromic shift in its absorption and emission spectra.