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BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAF[sup.V600E] mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAF[sup.V600E] has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC[sub.50] values in the 40–88 nM range. Selected compounds inhibited BRAF[sup.V600E] signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.

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 the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4 CRBN ) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4 CRBN . Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4 CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4 CRBN . Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4 CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins. The crystal structures of thalidomide and its derivatives bound to the E3 ligase subcomplex DDB1–CRBN are shown; these drugs are found to have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins. Thalidomide's dual mechanism of action Introduced in Europe in 1957 as a mild sedative, thalidomide was widely used in pregnant women as a treatment for morning sickness. This led to the birth of thousands of children with multiple defects and the drug was withdrawn in 1962. Since then thalidomide and its derivatives have emerged as effective treatments for the cancer multiple myeloma and the associated disorder 5q-dysplasia. The primary teratogenic target of thalidomide is cereblon (CRBN), part of E3 ubiquitin ligase complex CUL4–RBX1–DDB1–CRBN (CRL4 CRBN ). Here, Nicolas Thomä and colleagues present the crystal structure of DDB1–CRBN E3 ubiquitin ligase bound to thalidomide and to the related drugs lenalidomide and pomalidomide. The structure establishes the molecular mechanism underlying CRBN's enantioselective action. Further structure–function analysis reveals that these drugs have dual functions, interfering with the binding of certain cellular substrates to the E3 ligase but promoting the binding of others, thereby modulating the degradation of cellular proteins.

A multitude of new agents for the treatment of hematologic malignancies has been introduced over the past decade. Hematologists, infectious disease specialists, stem cell transplant experts, pulmonologists and radiologists have met within the framework of the European Conference on Infections in Leukemia (ECIL) to provide a critical state-of-the-art on infectious complications associated with immunotherapeutic and molecular targeted agents used in clinical routine. For brentuximab vedotin, blinatumomab, CTLA4- and PD-1/PD-L1-inhibitors as well as for ibrutinib, idelalisib, HDAC inhibitors, mTOR inhibitors, ruxolitinib, and venetoclax, a detailed review of data available until August 2018 has been conducted, and specific recommendations for prophylaxis, diagnostic and differential diagnostic procedures as well as for clinical management have been developed.

Thalidomide and the immunomodulatory drug, lenalidomide, are therapeutically active in hematological malignancies. The ubiquitously expressed E3 ligase protein cereblon (CRBN) has been identified as the primary teratogenic target of thalidomide. Our studies demonstrate that thalidomide, lenalidomide and another immunomodulatory drug, pomalidomide, bound endogenous CRBN and recombinant CRBN–DNA damage binding protein-1 (DDB1) complexes. CRBN mediated antiproliferative activities of lenalidomide and pomalidomide in myeloma cells, as well as lenalidomide- and pomalidomide-induced cytokine production in T cells. Lenalidomide and pomalidomide inhibited autoubiquitination of CRBN in HEK293T cells expressing thalidomide-binding competent wild-type CRBN, but not thalidomide-binding defective CRBN YW/AA . Overexpression of CRBN wild-type protein, but not CRBN YW/AA mutant protein, in KMS12 myeloma cells, amplified pomalidomide-mediated reductions in c-myc and IRF4 expression and increases in p21 WAF-1 expression. Long-term selection for lenalidomide resistance in H929 myeloma cell lines was accompanied by a reduction in CRBN, while in DF15R myeloma cells resistant to both pomalidomide and lenalidomide, CRBN protein was undetectable. Our biophysical, biochemical and gene silencing studies show that CRBN is a proximate, therapeutically important molecular target of lenalidomide and pomalidomide.

The crystal structure of pomalidomide Form I has been solved and refined using synchrotron X-ray powder diffraction data and optimized using density functional theory techniques. Pomalidomide Form I crystallizes in the space group P -1 (#2) with a = 7.04742(9), b = 7.89103(27), c = 11.3106(6) Å, α = 73.2499(13), β = 80.9198(9), γ = 88.5969(6)°, V = 594.618(8) Å 3 , and Z = 2. The crystal structure is characterized by the parallel stacking of planes parallel to the bc -plane. Hydrogen bonds link the molecules into double layers also parallel to the bc -plane. Each of the amine hydrogen atoms acts as a donor to a carbonyl group in an N–H⋯O hydrogen bond, but only two of the four carbonyl groups act as acceptors in such hydrogen bonds. Other carbonyl groups participate in C–H⋯O hydrogen bonds. The powder pattern has been submitted to ICDD® for inclusion in the Powder Diffraction File™ (PDF®).

In historical attempts to treat morning sickness, use of the drug thalidomide led to the birth of thousands of children with severe birth defects. Despite their teratogenicity, thalidomide and related IMiD drugs are now a mainstay of cancer treatment; however, the molecular basis underlying the pleiotropic biology and characteristic birth defects remains unknown. Here we show that IMiDs disrupt a broad transcriptional network through induced degradation of several C2H2 zinc finger transcription factors, including SALL4, a member of the spalt-like family of developmental transcription factors. Strikingly, heterozygous loss of function mutations in SALL4 result in a human developmental condition that phenocopies thalidomide-induced birth defects such as absence of thumbs, phocomelia, defects in ear and eye development, and congenital heart disease. We find that thalidomide induces degradation of SALL4 exclusively in humans, primates, and rabbits, but not in rodents or fish, providing a mechanistic link for the species-specific pathogenesis of thalidomide syndrome. Thalidomide was sold in the 1950s and 1960s as a sedative and anti-nausea medication for pregnant women suffering from morning sickness. Studies in mice and other animals had suggested thalidomide was safe and led some countries to allow the drug to be used in humans. By 1961, it became clear that thalidomide use by pregnant women led to serious birth defects, and the drug was removed from the market. By then, thalidomide had caused birth defects in over 10,000 babies, a tragedy that has been described as the biggest man-made medical disaster in human history. It led many countries to adopt tougher standards for drug safety. Thalidomide and similar drugs are now used with great success to treat leprosy and various blood cancers. But questions remain about exactly how the drugs work and how they cause birth defects like shortened arms and legs. Previous studies have shown that thalidomide binds to a protein called cereblon, which marks other proteins for destruction and removal from the cell. Thalidomide hijacks cereblon and causes it to tag the wrong proteins. To learn more about how thalidomide causes birth defects, Donovan et al. treated human embryonic stem cells and cancer cells with thalidomide and related drugs. Analyzing the proteins inside the cells revealed that the drugs caused dramatic reductions in the amount of a protein called SALL4, which is essential for limb development. It was already known that mutations in the gene that produces SALL4 cause two conditions called Duane Radial Ray syndrome and Holt-Oram syndrome. Both conditions can result in birth defects like those seen in babies exposed to thalidomide. As well as showing that thalidomide-hijacked cereblon marks SALL4 for destruction, Donovan et al. also reveal why mice do not develop birth defects when exposed to thalidomide. This is because genetic differences make the mouse cereblon proteins unable to tag SALL4. Researchers could now build on these results to develop safer versions of thalidomide that do not target SALL4 while still successfully treating leprosy and cancers.

Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein–protein interactions. Proteins in a cell need to interact with each other to perform the many tasks required for organisms to thrive. A technique called proximity biotinylation helps scientists to pinpoint the identity of the proteins that partner together. It relies on attaching an enzyme (either BioID or TurboID) to a protein of interest; when a partner protein comes in close contact with this construct, the enzyme can attach a chemical tag called biotin to it. The tagged proteins can then be identified, revealing which molecules interact with the protein of interest. Although BioID and TurboID are useful tools, they have some limitations. Experiments using BioID take more than 16 hours to complete and require high levels of biotin to be added to the cells. TurboID is more active than BioID and is able to label proteins within ten minutes. However, under certain conditions, it is also more likely to be toxic for the cell, or to make mistakes and tag proteins that do not interact with the protein of interest. To address these issues, Kido et al. developed AirID, a new enzyme for proximity biotinylation. Experiments were then conducted to test how well AirID would perform, using proteins of interest whose partners were already known. These confirm that AirID was able to label partner proteins in human cells; compared with TurboID, it was also less likely to mistakenly tag non-partners or to kill the cells, even over long periods. The results by Kido et al. demonstrate that AirID is suitable for proximity biotinylation experiments in cells. Unlike BioID and TurboID, the enzyme may also have the potential to be used for long-lasting experiments in living organisms, since it is less toxic for cells over time.

Thalidomide, originally developed as a sedative drug, causes multiple defects due to severe teratogenicity, but it has been re-purposed for treating multiple myeloma, and derivatives such as lenalidomide and pomalidomide have been developed for treating blood cancers. Although the molecular mechanisms of thalidomide and its derivatives remained poorly understood until recently, we identified cereblon (CRBN), a primary direct target of thalidomide, using ferrite glycidyl methacrylate (FG) beads. CRBN is a ligand-dependent substrate receptor of the E3 ubiquitin ligase complex cullin-RING ligase 4 (CRL4CRBN). When a ligand such as thalidomide binds to CRBN, it recognizes various ‘neosubstrates’ depending on the shape of the ligand. CRL4CRBN binds many neosubstrates in the presence of various ligands. CRBN has been utilized in a novel protein knockdown technology named proteolysis targeting chimeras (PROTACs). Heterobifunctional molecules such as dBET1 are being developed to specifically degrade proteins of interest. Herein, we review recent advances in CRBN research.