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.

The WWOX tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically-relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically-distinct E66/Y85 duo at structurally-equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, introduction of E66R/Y85W double-substitution within the WW2 domain not only results in gain-of-function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease.

No abstract

YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery (ITC) and circular dichroism (CD) in combination with molecular modeling (MM) and molecular dynamics (MD), we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, non-consensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II (PPII) helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a non-bulky and flexible glycine one-residue C-terminal to the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, arguing that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

The link between breast cancer and estrogen receptor (ER) is well established. The ER is a hormone-inducible transcription factor that, upon binding to its ligand, regulates the expression of a variety of genes mainly involved in cell proliferation and differentiation. Coactivators are proteins recruited by the hormone-activated receptor, which allow or enhance the ER transactivation functions by acting as chromatin remodeling enzymes or adaptors between ER and the transcriptional machinery. Our laboratory has previously identified the WW-domain binding protein-2 (WBP-2) as a bona fide coactivator of ER. However, the molecular mechanism underlying WBP-2 coactivation function was not clear yet. In this study, we explore and identify the mechanism by which WBP-2 acts as coactivator of ER. Our data show that WBP-2 is involved in the regulation of ER target genes, and its expression is required for the proper expression of some ER target genes.To clarify the molecular mechanism by which WBP-2 regulates ER function, we performed chromatin immunoprecipitation assays. We demonstrate here that WBP-2 binds to the ER target gene promoter pS2 promoter and is required for the binding of the phosphorylated form of RNA polymerase II (associated with active transcription/elongation) to the same promoter. Furthermore, we also show that WBP-2 is essential for the recruitment of the histone acetyl transferase p300, an important chromatin modifier enzyme and for histone acetylation at the same target region. Collectively, our data indicate that WBP-2 enhances ER transactivation function at certain genes by facilitating the recruitment and/or the stabilization of a histone modifier enzyme that favors a relaxed chromatin structure, permissive of transcription. V C 2012 IUBMB Life, 65(1): [76][77][78][79][80][81][82][83][84] 2013

3,4,5-Trisubstituted-1,2,4-4H-triazoles (TTs) have recently been identified as a new class of potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Two series of triazoles have been studied, one of which was also screened against the Y188L mutant. A computational strategy based on molecular docking studies followed by comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) has been used to elucidate the atomic details of the RT/TT interactions and to identify the most important features impacting the TT antiretroviral activity. Two 3D-QSAR CoMFA and CoMSIA models were derived, using the TT pEC₅₀ values measured against wild-type (WT) HIV-1 (model A) and the Y188L mutant form (model B), respectively, as the dependent variable. The final model A CoMSIA (r(ncv)²  = 0.97, r(cv)²  = 0.89, SEE = 0.314, and r(pred)²  = 0.82) and model B CoMSIA (r(ncv)²  = 0.91, r(cv)²  = 0.61, SEE = 0.236, and r(pred)²  = 0.73) analyses were more predictive. The results allowed us to obtain useful information for the design of new compounds with improved potency towards WT HIV-1 or that are potentially active against the Y188L mutant.

No abstract

No abstract