Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

DNA breakage is intimately associated with meiotic recombination in the fission yeast Schizosaccharomyces pombe. Sites of prominent DNA breakage were found approximately 25 to approximately 200 kb apart in the genomic regions surveyed. We examined in detail a 501 kb region of chromosome I and found six sites, or tight clusters of sites, at which approximately 2%-11% of the DNA accumulated breaks in a rad50S mutant. In contrast to the discrete, widely spaced distribution of prominent break sites, recombination in this region was more uniformly distributed (0.7-1.6 cM/10 kb) whether the genetic interval tested contained no, one, or more such sites. We infer that although recombination depends upon DNA breakage, recombination often occurs remote from these sites (tens of kilobases away); we discuss mechanisms by which this may occur.

During meiosis DNA double-strand breaks initiate recombination in the distantly related budding and fission yeasts and perhaps in most eukaryotes. Repair of broken meiotic DNA is essential for formation of viable gametes. We report here distinct but overlapping sets of proteins in these yeasts required for formation and repair of double-strand breaks. Meiotic DNA breakage in Schizosaccharomyces pombe did not require Rad50 or Rad32, although the homologs Rad50 and Mre11 are required in Saccharomyces cerevisiae ; these proteins are required for meiotic DNA break repair in both yeasts. DNA breakage required the S. pombe midmeiosis transcription factor Mei4, but the structurally unrelated midmeiosis transcription factor Ndt80 is not required for breakage in S. cerevisiae. Rhp51, Swi5, and Rad22 ϩ Rti1 were required for full levels of DNA repair in S. pombe, as are the related S. cerevisiae proteins Rad51, Sae3, and Rad52. Dmc1 was not required for repair in S. pombe, but its homolog Dmc1 is required in the well-studied strain SK1 of S. cerevisiae. Additional proteins required in one yeast have no obvious homologs in the other yeast. The occurrence of conserved and nonconserved proteins indicates potential diversity in the mechanism of meiotic recombination and divergence of the machinery during the evolution of eukaryotes. I N most eukaryotes homologous recombination ocobserved more recently in a second organism, the distantly related fission yeast Schizosaccharomyces pombe (Cercurs at high levels during meiosis to aid the proper segregation of homologs at the first meiotic division vantes et al. 2000). There are two lines of indirect evidence for meiotic ds breaks in other organisms. First, and to increase genetic diversity among gametes, the products of meiosis. The two meiotic cell divisions revarious eukaryotes encode homologs of the Spo11 protein, which is essential for meiotic DNA ds break formaduce the diploid number of chromosomes in the precursor cells to the haploid number in the gametes. The tion in S. cerevisiae (Keeney et al. 1997). Where tested in other organisms, these proteins are essential for meiotic general mechanism of reductional segregation of homologs is highly conserved: in most eukaryotes recombinarecombination or viable gamete formation (Keeney 2001), and in S. pombe the homolog, called Rec12, is tion between homologs provides a physical connection between them that imparts tension when the homologs also essential for meiotic DNA ds break formation (Cervantes et al. 2000). Second, in mice a modified histone, are properly arranged to segregate to opposite poles of the cell (Nicklas 1997). In the absence of recombina-␥-H2AX, thought to associate with chromatin specifically near ds breaks, appears as foci on meiotic chromotion, homologs frequently missegregate, resulting in aneuploid gametes; the subsequent zygotic progeny are somes at the time expected for recombination; these foci are Spo11 dependent (Rogakou et al. 1998; Mahafrequently sick or dead, underscoring the importance of un...

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Key Points Blockmirs are designed against the miR-27 binding site in VE-cadherin and display restricted specificity. Blockmirs regulate VE-cadherin and endothelial cell junctions, inhibit edema, and promote angiogenesis associated with ischemia.