Structure-activity relationships of thiadiazole agonists of the human secretin receptor.

Agonists of the secretin receptor have potential applications for diseases of the cardiovascular, gastrointestinal, and metabolic systems, yet no clinically-active non-peptidyl agonists of this receptor have yet been developed. In the current work, we have identified a new small molecule lead compound with this pharmacological profile. We have prepared and characterized a systematic structure-activity series around this thiadiazole scaffold to better understand the molecular determinants of its activity. We were able to enhance the in vitro activity and to maintain the specificity of the parent compound. We found the most active candidate to be quite stable in plasma, although it was metabolized by hepatic microsomes. This chemical probe should be useful for in vitro studies and needs to be tested for in vivo pharmacological activity. This could be an important lead toward the development of a first-in-class orally active agonist of the secretin receptor, which could be useful for multiple disease states.

PD-1 derived CA-170 is an oral immune checkpoint inhibitor that exhibits preclinical anti-tumor efficacy.

Small molecule immune checkpoint inhibitors targeting PD-1 and other pathways may offer advantages including ease of dosing, ability to manage immune-related adverse events (irAEs) due to their shorter pharmacokinetic exposure and opportunity to target more than one pathway for improving efficacy. Here we describe the identification and characterization of CA-170, an amino acid inspired small molecule inhibitor of PD-L1 and VISTA derived from the interface of PD-1 and PD-L1. CA-170 exhibited potent rescue of proliferation and effector functions of T cells inhibited by PD-L1/L2 and VISTA with selectivity over other immune checkpoint proteins as well as a broad panel of receptors and enzymes. Observed blocking of PD-L1 signaling and binding to PD-L1 in the cellular context without preventing the assembly of PD-1:PD-L1 complex support the formation of a defective ternary complex as the mechanism of action of CA-170. Oral administration of CA-170 resulted in increased proliferation and activation of T cells in the tumor, and significant anti-tumor efficacy in a number of immunocompetent mouse tumor models either as a single agent or in combination with approved therapeutics. These results prompted the advancement of CA-170 to human clinical trials.

A Rationally Designed Peptide Antagonist of the PD-1 Signaling Pathway as an Immunomodulatory Agent for Cancer Therapy.

Pioneering success of antibodies targeting immune checkpoints such as PD-1 and CTLA4 has opened novel avenues for cancer immunotherapy. Along with impressive clinical activity, severe immune-related adverse events (irAE) due to the breaking of immune self-tolerance are becoming increasingly evident in antibody-based approaches. As a strategy to better manage severe adverse effects, we set out to discover an antagonist targeting PD-1 signaling pathway with a shorter pharmacokinetic profile. Herein, we describe a peptide antagonist NP-12 that displays equipotent antagonism toward PD-L1 and PD-L2 in rescue of lymphocyte proliferation and effector functions. In preclinical models of melanoma, colon cancer, and kidney cancers, NP-12 showed significant efficacy comparable with commercially available PD-1-targeting antibodies in inhibiting primary tumor growth and metastasis. Interestingly, antitumor activity of NP-12 in a preestablished CT26 model correlated well with pharmacodynamic effects as indicated by intratumoral recruitment of CD4 and CD8 T cells, and a reduction in PD-1+ T cells (both CD4 and CD8) in tumor and blood. In addition, NP-12 also showed additive antitumor activity in preestablished tumor models when combined with tumor vaccination or a chemotherapeutic agent such as cyclophosphamide known to induce "immunologic cell death." In summary, NP-12 is the first rationally designed peptide therapeutic targeting PD-1 signaling pathways exhibiting immune activation, excellent antitumor activity, and potential for better management of irAEs.

ODM-203, a Selective Inhibitor of FGFR and VEGFR, Shows Strong Antitumor Activity, and Induces Antitumor Immunity.

Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC50 6-35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC50 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50-150 nmol/L). In vivo, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.

Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): their expression during early embryonic development.

BACKGROUND: Current literature and our previous results on expression patterns of oocyte-specific genes and transcription factors suggest a global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). MicroRNAs (miRNAs) are small, non-coding regulatory RNAs (19-23 nucleotides) that regulate gene expression by guiding target mRNA cleavage or translational inhibition. These regulatory RNAs are potentially involved in the degradation of maternally inherited mRNAs during early embryogenesis. RESULTS: To identify miRNAs that might be important for early embryogenesis in rainbow trout, we constructed a miRNA library from a pool of unfertilized eggs and early stage embryos. Sequence analysis of random clones from the library identified 14 miRNAs, 4 of which are novel to rainbow trout. Real-time PCR was used to measure the expression of all cloned miRNAs during embryonic development. Four distinct expression patterns were observed and some miRNAs showed up-regulated expression during EGA. Analysis of tissue distribution of these miRNAs showed that some are present ubiquitously, while others are differentially expressed among different tissues. We also analyzed the expression patterns of Dicer, the enzyme required for the processing of miRNAs and Stat3, a transcription factor involved in activating the transcription of miR-21. Dicer is abundantly expressed during EGA and Stat3 is up-regulated before the onset of EGA. CONCLUSION: This study led to the discovery of 14 rainbow trout miRNAs. Our data support the notion that Dicer processes miRNAs and Stat3 induces expression of miR-21 and possibly other miRNAs during EGA. These miRNAs in turn guide maternal mRNAs for degradation, which is required for normal embryonic development.

Molecular characterization and expression profiles of cyclin B1, B2 and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout (Oncorhynchus mykiss).

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1, CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes, but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expression patterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes and spermotocytes.

Debio 0617B Inhibits Growth of STAT3-Driven Solid Tumors through Combined Inhibition of JAK, SRC, and Class III/V Receptor Tyrosine Kinases.

Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non-small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334-43. ©2016 AACR.

Identification of OORP-T, a novel oocyte-specific gene encoding a protein with a conserved oxysterol binding protein domain in rainbow trout.

Genes specifically expressed in oocytes are important for the development of oocytes and early embryos. By analyzing expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified a novel EST sequence that does not show homology to any sequences in the GenBank. Analysis of tissue distribution by RT-PCR revealed that this gene was only expressed in unfertilized oocytes. Sequencing of the EST clone identified a cDNA of 3,163 bp. Northern blot analysis showed the novel gene has a single transcript of 3.4 kb. Additional 5' sequence was obtained by 5' RACE, extending the novel cDNA to 3,333 bp. Analysis of the full-length cDNA identified an open reading frame (ORF) encoding a protein of 564 amino acids. The novel protein contains a conserved oxysterol binding protein (OSBP) domain at the C terminus that is characteristic of OSBP-related proteins (ORPs) implicated in lipid metabolism. Therefore, we named the novel gene as Oocyte-specific Oxysterol binding protein Related-Protein of Trout (OORP-T). In situ hybridization showed that the OORP-T mRNA appears to be confined to the cytoplasm of vitellogenic oocytes. Transcription of OORP-T appears to start during pre-vitellogenesis and increases steadily, reaching its peak in the late vitellogenic stage. OORP-T transcript is abundantly present in unfertilized eggs but the level drops significantly in day 2 embryos and continues to decline in day 7 embryos after which it remains low. We propose that OORP-T may play an important role in the utilization of yolk-derived lipid products during oocyte development and early stages of embryonic development in rainbow trout.