Total chemical synthesis of two Conus-derived peptides, conantokin-G (con-G), a 17-residue polypeptide containing five residues of gamma-carboxyglutamic acid (Gla), and conantokin-T (con-T), a 21-residue polypeptide possessing four residues of Gla, was accomplished. Calcium binding isotherms were obtained for each peptide, and these differed considerably from each other. The binding isotherm for con-G was complex and could only be fit to degenerate models involving multiple Ca2+ binding sites. The data for Ca2+ binding to con-T was uniquely fit to a simple one-site model. In the case of con-G, circular dichroism (CD) studies revealed a polypeptide without observable alpha-helicity in the absence of Ca2+ and a dramatic shift to a high degree of alpha-helix at saturating Ca2+ concentrations. In contrast, apo-con-T possessed significant alpha-helical structure, and saturation with Ca2+ produced a less substantial change in its alpha-helical content. Titrations with Ca2+ of the change in alpha-helical content of con-T produced a C50 value for Ca2+ that was essentially the same as its Kd from direct binding studies, demonstrating that occupancy of the single macroscopic binding site resulted in the conformational change. Similar titrations with con-G provided a C50 value in concert with the Kd for binding of Ca2+ to this peptide. Moreover, in agreement with these particular Ca(2+)-induced structural changes, gel filtration analyses demonstrated significantly reduced hydrodynamic volumes of both of these polypeptides after saturation of their apo forms with Ca2+, with con-G showing a more pronounced change than con-T. One-dimensional H-NMR spectra showed both line broadening and changes in chemical shifts of several peptide amide proton resonances after addition of Ca2+ to con-G, again suggestive of a large Ca(2+)-induced conformational change in this polypeptide. A derivative of con-G was synthesized with all amino acids present in the D-configuration (D-con-G). This variant peptide displayed Ca2+ binding isotherms nearly identical to those of con-G and underwent a Ca(2+)-induced conformational change very similar to that of con-G. Intracranial injections of con-G and con-T in young (< 2 weeks) and older (3-4 weeks) mice produced the expected "sleep-like" and hyperactive effects, respectively. The variant, D-con-G, was inactive in these assays. These studies demonstrate that synthetic con-G and con-T possess their expected bioactivities and undergo large and defined conformational alterations in the presence of Ca2+. We propose that binding of Ca2+ to these polypeptides contributes to their ability to adopt a defined conformation, and this divalent cation-dependent conformation is necessary for their neuroactivities.

The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of synergy between ABBV-075 and these agents. The / activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. .

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