Normal peripheral nerve myelination depends on Schwann cell-basal lamina interactions. An important component of Schwann cell basal lamina is laminin--predominantly laminins 2 and 4. Mutations in the alpha 2 chain common to these two isoforms are associated with dysmyelination in mouse (dy) and man (congenital muscular dystrophy). Thus, laminin 2 and 4 receptors are also likely to be important for myelin formation. Several laminin 2/4 receptors are detected at the basal lamina surface of myelin-forming Schwann cells, namely, alpha 6 beta 4 and alpha 6 beta 1 integrins and dystroglycan. The evidence linking these receptors to myelination is suggestive, but not conclusive. Genetic studies have not yet confirmed a role for these molecules in myelin formation. Natural or targeted inactivation of alpha 6, beta 4, and beta 1 integrins and of dystroglycan have profound effects on other tissues causing embryonic or perinatal death before myelination. Therefore, to conditionally inactivate these receptors specifically in myelin-forming Schwann cells, we have constructed and initially characterized a P0-Cre transgene that activates Cre-mediated recombination of loxP-containing genes in peripheral nerve.

Background: HCN2 and HCN4 respond to cAMP, whereas HCN1 does not. Results: The C-linker plus CNBD of HCN2 and HCN4 show cAMP-induced tetramerization, whereas that of HCN1 contains prebound cAMP and is tetrameric. Conclusion: HCN1 does not respond to the addition of cAMP because its CNBD contains cAMP already. Significance: Tetramerization of the C terminus controls ligand gating in HCN channels.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.

CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.