In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins.

Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.

The results confirm the usefulness of synthetic heterotrimeric collagenous peptides in the folded and unfolded state as mimics of the natural substrates collagen and gelatin, respectively, to gain a better a insight into the proteolytic mechanisms of matrix metalloproteinases.

A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-P'+/t0) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)s triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triplestranded cystine peptide. While the single chains showed no teodeney to self-association into triple helices, the heterotrimer (cxltz2~xl') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tin) of

Collagenous peptides containing the collagenase cleavage site α1(772−784) and α2(772−784) of collagen type I were synthesized and assembled into heterotrimers via regioselective C-terminal interchain-disulfide bridging in a defined α1α2α1‘ staggered register of the three peptide strands. Various approaches were attempted to induce and stabilize the collagen-characteristic triple-helical fold even in the sequence portion of the collagenase cleavage site with its weak triple-helix propensity. By N-terminal chain elongation with (Gly-Pro-Hyp) n tripeptide repeats, particularly with n = 5, and in an even more pronounced manner, by incorporation of an additional tripeptide repeat adjacent to the cystine knot, a collagenous heterotrimer was obtained which was found to exhibit dichroic properties fully consistent with the triple-helical fold. Thermal denaturation revealed a remarkable stability with a melting temperature of 41 °C. Although the complex cystine knot of natural collagen was reduced in these synthetic heterotrimers to two interchain-disulfide bridges, it showed not only the expected entropic contribution to the refolding process by keeping the three chains assembled, but more importantly a triple-helix nucleation was induced. In fact, temperature jump experiments clearly revealed two-phase refolding kinetics very similar to those of the disulfide-bridged natural collagen fragment of Col 1−3, where refolding without nucleation difficulty was obtained followed by a slower process dominated by the cis → trans isomerization for triple-helix propagation. These results would indicate that even the simplified artificial cystine knot is capable of aligning the three peptide chains in the defined α1α2α1‘ one-residue shift register. Moreover, the synthetic heterotrimers were cleaved by interstitial collagenases in a single cut through all three chains without release of intermediates during the relatively slow enzymatic digestion process. This observation confirms that, with the de novo designed heterotrimers, functional collagen epitopes were mimicked in highly efficient manner; it also strongly suggests that the preselected α1α2α1‘ register may indeed represent the correct staggered alignment of the α subunits at least in collagen type I.

(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERR␥, estrogen receptor (ER) ␣, and ER␤. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERR␥ but resembles the situation for ER␣ (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERR␣ and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRAS G12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique, covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRAS G12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRAS G12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with SHP2, MEK or CDK4/6 inhibitors. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRAS G12C-mutated tumors.