Discovery of diverse small molecule chemotypes with cell-based PKD1 inhibitory activity

Protein kinase D (PKD) is a novel family of serine/threonine kinases regulated by diacylglycerol, which is involved in multiple cellular processes and various pathological conditions. The limited number of cell-active, selective inhibitors has historically restricted biochemical and pharmacological studies of PKD. We now markedly expand the PKD1 inhibitory chemotype inventory with eleven additional novel small molecule PKD1 inhibitors derived from our high throughput screening campaigns. The in vitro IC(50)s for these eleven compounds ranged in potency from 0.4 to 6.1 μM with all of the evaluated compounds being competitive with ATP. Three of the inhibitors (CID 1893668, (1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one; CID 2011756, 5-(3-chlorophenyl)-N-[4-(morpholin-4-ylmethyl)phenyl]furan-2-carboxamide; CID 5389142, (6Z)-6-[4-(3-aminopropylamino)-6-methyl-1H-pyrimidin-2-ylidene]cyclohexa-2,4-dien-1-one) inhibited phorbol ester-induced endogenous PKD1 activation in LNCaP prostate cancer cells in a concentration-dependent manner. The specificity of these compounds for PKD1 inhibitory activity was supported by kinase assay counter screens as well as by bioinformatics searches. Moreover, computational analyses of these novel cell-active PKD1 inhibitors indicated that they were structurally distinct from the previously described cell-active PKD1 inhibitors while computational docking of the new cell-active compounds in a highly conserved ATP-binding cleft suggests opportunities for structural modification. In summary, we have discovered novel PKD1 inhibitors with in vitro and cell-based inhibitory activity, thus successfully expanding the structural diversity of small molecule inhibitors available for this important pharmacological target.     

Physiological and biochemical characterization of NERICA‐L‐44: a novel source of heat tolerance at the vegetative and reproductive stages in rice

The predicted increase in the frequency and magnitude of extreme heat spikes under future climate can reduce rice yields significantly. Rice sensitivity to high temperatures during the reproductive stage is well documented while the same during the vegetative stage is more speculative. Hence, to identify and characterize novel heat‐tolerant donors for both the vegetative and reproductive stages, 71 rice accessions, including approximately 75% New Rice for Africa (NERICAs), were phenotyped across field experiments during summer seasons in Delhi, India, and in a controlled environment study at International Rice Research Institute , Philippines. NERICA‐L‐44 (NL‐44) recorded high seedling survival (52%) and superior growth and greater reproductive success exposed to 42.2°C (sd ± 2.3) under field conditions. NL‐44 and the heat‐tolerant check N22 consistently displayed lower membrane damage and higher antioxidant enzymes activity across leaves and spikelets. NL‐44 recorded 50–60% spikelet fertility, while N22 recorded 67–79% under controlled environment temperature of 38°C (sd ±1.17), although both had about 87% fertility under extremely hot field conditions. N22 and NL‐44, exposed to heat stress (38°C), had similar pollen germination percent and number of pollen tubes reaching the ovary. NL‐44 maintained low hydrogen peroxide production and non‐photochemical quenching (NPQ) with high photosynthesis while N22 avoided photosystem II damage through high NPQ under high‐temperature stress. NL‐44 with its reproductive stage resilience to extreme heat stress, better antioxidant scavenging ability in both vegetative tissue and spikelets and superior yield and grain quality is identified as a novel donor for increasing heat tolerance at both the vegetative and reproductive stages in rice.     

Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro

Background  

The initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2.