The discovery of (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile, a potent and selective agonist of human transient receptor potential cation channel subfamily m member 5 (TRPM5) and evaluation of as a potential gastrointestinal prokinetic agent.

This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.

PIM kinases are progression markers and emerging therapeutic targets in diffuse large B-cell lymphoma.

BACKGROUND: PIM serine/threonine kinases are often highly expressed in haematological malignancies. We have shown that PIM inhibitors reduced the survival and migration of leukaemic cells. Here, we investigated PIM kinases in diffuse large B-cell lymphoma (DLBCL) biopsy samples and DLBCL cell lines. METHODS: Immunohistochemical staining for PIM kinases and CXCR4 was performed on tissue microarrays from a cohort of 101 DLBCL cases, and the effects of PIM inhibitors on the survival and migration of DLBCL cell lines were determined. RESULTS: PIM1 expression significantly correlated with the activation of signal transducer and activator of transcription (STAT) 3 and 5, P-glycoprotein expression, CXCR4-S339 phosphorylation, and cell proliferation. Whereas most cases exhibited cytoplasmic or cytoplasmic and nuclear PIM1 and PIM2 expression, 12 cases (10 of the non-germinal centre DLBCL type) expressed PIM1 predominately in the nucleus. Interestingly, nuclear expression of PIM1 significantly correlated with disease stage. Exposure of DLBCL cell lines to PIM inhibitors modestly impaired cellular proliferation and CXCR4-mediated migration. CONCLUSION: This work demonstrates that PIM expression in DLBCL is associated with activation of the JAK/STAT signalling pathway and with the proliferative activity. The correlation of nuclear PIM1 expression with disease stage and the modest response to small-molecule inhibitors suggests that PIM kinases are progression markers rather than primary therapeutic targets in DLBCL.

Correction: Interleukin-22-deficiency and microbiota contribute to the exacerbation of Toxoplasma gondii-induced intestinal inflammation.

The original version of this Article omitted the author Dr Mathias Chamaillard from the l'Institut de Pasteur, Lille, France. This has been corrected in both the PDF and HTML versions of the Article.

Interleukin-22-deficiency and microbiota contribute to the exacerbation of Toxoplasma gondii-induced intestinal inflammation.

Upon oral infection with Toxoplasma gondii cysts (76 K strain) tachyzoites are released into the intestinal lumen and cross the epithelial barrier causing damage and acute intestinal inflammation in C57BL/6 (B6) mice. Here we investigated the role of microbiota and IL-22 in T.gondii-induced small intestinal inflammation. Oral T.gondii infection in B6 mice causes inflammation with IFNγ and IL-22 production. In IL-22-deficient mice, T.gondii infection augments the Th1 driven inflammation. Deficiency in either IL-22bp, the soluble IL-22 receptor or Reg3γ, an IL-22-dependent antimicrobial lectin/peptide, did not reduce inflammation. Under germ-free conditions, T.gondii-induced inflammation was reduced in correlation with parasite load. But intestinal inflammation is still present in germ-free mice, at low level, in the lamina propria, independently of IL-22 expression. Exacerbated intestinal inflammation driven by absence of IL-22 appears to be independent of IL-22 deficiency associated-dysbiosis as similar inflammation was observed after fecal transplantation of IL-22-/- or WT microbiota to germ-free-WT mice. Our results suggest cooperation between parasite and intestinal microbiota in small intestine inflammation development and endogenous IL-22 seems to exert a protective role independently of its effect on the microbiota. In conclusion, IL-22 participates in T.gondii induced acute small intestinal inflammation independently of microbiota and Reg3γ.

CXCR4-SERINE339 regulates cellular adhesion, retention and mobilization, and is a marker for poor prognosis in acute myeloid leukemia.

The CXCR4 receptor is a major regulator of hematopoietic cell migration. Overexpression of CXCR4 has been associated with poor prognosis in acute myelogenous leukemia (AML). We have previously shown that ligand-mediated phosphorylation of the Serine339 (CXCR4-S339) residue of the intracellular domain by PIM1 is implicated in surface re-expression of this receptor. Here, we report that phosphorylation of CXCR4-S339 in bone marrow (BM) biopsies correlated with poor prognosis in a cohort of AML patients. To functionally address the impact of CXCR4-S339 phosphorylation, we generated cell lines-expressing CXCR4 mutants that mimic constitutive phosphorylation (S339E) or abrogate phosphorylation (S339A). Whereas the expression of CXCR4 significantly increased, both CXCR4-S339E and the CXCR4-S339A mutants significantly reduced the BM homing and engraftment of Kasumi-1 AML cells in immunodeficient mice. In contrast, only expression of the CXCR4-S339E mutant increased the BM retention of the cells and resistance to cytarabine treatment, and impaired detachment capacity and AMD3100-induced mobilization of engrafted leukemic cells. These observations suggest that the poor prognosis in AML patients displaying CXCR4-S339 phosphorylation can be the consequence of an increased retention to the BM associated with an enhanced chemoresistance of leukemic cells. Therefore, CXCR4-S339 phosphorylation could serve as a novel prognostic marker in human AML.

The frequency of the equine cerebellar abiotrophy mutation in non-Arabian horse breeds.

REASONS FOR PERFORMING STUDY: A putative mutation causative of cerebellar abiotrophy (CA), a genetic defect found almost exclusively in Arabian horses, was recently identified. OBJECTIVES AND HYPOTHESIS: To investigate the presence of the CA mutation in breeds other than Arabian and ascertain whether the mutation had been introduced into these breeds by Arabian ancestry. The CA mutation is present in breeds of horses with Arabian ancestry. METHODS: Allele-specific PCR was used to genotype 1845 non-Arabian horses for the CA mutation. For those breeds in which at least one carrier was identified, an additional 266 horses were genotyped to determine the frequency of the CA allele. Cerebellar abiotrophy carriers were further genotyped for a haplotype segregating with CA in Arabians. RESULTS: At least one CA carrier was identified in 3 breeds and the frequency of the CA allele calculated: Bashkir Curly Horses (2.8%), Trakehners (0.68%) and Welsh ponies (0.33%). Based on pedigree and haplotype analysis, CA was introduced into these breeds by Arabian ancestry. The Trakehner and Welsh pony carriers were at least half-Arabian, while the Bashkir Curly horses appeared to have had the CA allele introduced by a single Arabian stallion used for developing the breed in the 1960s. CONCLUSIONS: The CA mutation is present in breeds of horses that allow crossbreeding with Arabian horses and in breeds that have used Arabians as foundation stock during their development. POTENTIAL RELEVANCE: Breeds that allow registration of horses with Arabian ancestry should have any Arabian breeding stock tested for the mutation and breeds descended from Arabian ancestry should pursue genetic testing of breeding stock to prevent the occurrence of affected foals.

Functional characterization of high levels of meningioma 1 as collaborating oncogene in acute leukemia.

Retroviral expression of leukemogenic oncogenes in the murine hematopoietic system is essential but not sufficient to induce acute leukemia. Proviral integration-mediated elevated expression of the meningioma 1 (MN1) oncogene suggested MN1 acting as cooperating event in mixed-lineage leukemia 1 (MLL) and eleven nineteen leukemia (ENL)-induced murine leukemia. Indeed, co-expression of MN1 with MLL-ENL enhanced transformation in vivo, and resulted in a significantly reduced latency for induction of an aggressive acute leukemia when compared with MN1 or MLL-ENL alone. In addition, co-expression of MN1 increased the granulocyte macrophage progenitor cell population with leukemia-initiating properties as shown in secondary transplantation experiments. Gene expression profiling experiments identified putative downstream MN1 targets, of which FMS-like tyrosine kinase 3 (FLT3) and CD34 were upregulated in both MN1-overexpressing murine leukemias and in pediatric acute leukemias with high MN1 levels. Interestingly, small interfering RNA (siRNA)-mediated MN1 knockdown resulted in cell cycle arrest and impaired clonogenic growth of human leukemia cell lines with high MN1 levels. Our work shows for the first time that high MN1 levels are important for the growth of leukemic cells, and that increased MN1 expression can synergize with MLL-ENL and probably other transforming fusion genes in leukemia induction through a distinct gene expression program that is able to expand the leukemia-initiating cell population.