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.

Driven by the ever-increasing pace of drug discovery and the need to push the boundaries of unexplored chemical space, medicinal chemists are routinely turning to unusual strained bioisosteres such as bicyclo[1.1.1]pentane, azetidine, and cyclobutane to modify their lead compounds. Too often, however, the difficulty of installing these fragments surpasses the challenges posed even by the construction of the parent drug scaffold. This full account describes the development and application of a general strategy where spring-loaded, strained C–C and C–N bonds react with amines to allow for the “any-stage” installation of small, strained ring systems. In addition to the functionalization of small building blocks and late-stage intermediates, the methodology has been applied to bioconjugation and peptide labeling. For the first time, the stereospecific strain-release “cyclopentylation” of amines, alcohols, thiols, carboxylic acids, and other heteroatoms is introduced. This report describes the development, synthesis, scope of reaction, bioconjugation, and synthetic comparisons of four new chiral “cyclopentylation” reagents.

The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.

Limited tryptic digestion of the pyruvate dehydrogenase complex of Escherichia coli or its dihydrolipoyl transacetylase core cleaves the trypsin-sensitive transacetylase subunits into two large fragments, A (lipoyl domain) The pyruvate dehydrogenase complex from Escherichia coli is organized about a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase are joined by noncovalent bonds (1). The appearance of the transacetylase in the electron microscope is that of a cube, and its design appears to be based on octahedral (432) symmetry (2). The transacetylase consists of 24 apparently identical polypeptide chains, and each transacetylase chain apparently contains two (3-5) or possibly three (6) covalently bound lipoyl moieties. Lipoic acid is bound in amide linkage to the E-amino group of a lysyl residue (7). The lipoyl moiety is thought to rotate among the catalytic centers of the complex (8). This communication reports the results of studies of limited tryptic digestion of the E. coli dihydrolipoyl transacetylase in which novel aspects of its subunit structure were revealed. It appears that the native transacetylase subunit contains a compact domain of Mr -29,600 that possesses all the intersubunit binding sites as well as the catalytic site. To this domain is attached a flexible extension of Mr -31,600 containing the lipoyl moieties. MATERIALS AND METHODSDL-[2-3H]Lipoic acid (30,000-44,000 cpm/,ug) was synthesized by metalation of lipoic acid at C-2 (9) followed by treatment with 3H20 (J. R. Butler and L. J. Reed, unpublished data). N-Ethyl[2,3-14C]maleimide was purchased from Amersham/ Searle. L-(1-Tosylamido-2-phenyl)ethyl chloromethyl ketone-treated trypsin was obtained from Worthington, and soybean trypsin inhibitor (type 1-S) was from Sigma.Pyruvate dehydrogenase complex containing [2-3H]lipoic acid covalently bound to the dihydrolipoyl transacetylase component was isolated from E. coli (Crookes strain) grown in the presence of 102-125 ,ug of DL-[2-3H]lipoic acid per liter of culture medium as described (8, 10 Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was performed in 7.5% acrylamide gels by the procedure of Weber and Osborn (12). The samples were heated at 1000 C for 3-5 min. MrS were estimated from these gels by use of suitable marker proteins (10). For radioactivity measurement the gels were sliced into 1-or 2-mm sections, and the slices were dissolved in 30% hydrogen peroxide. Alternatively, N-N'-diallyl tartardiamide (Eastman) was used instead of methylene bisacrylamide as the crosslinking agent, and the gel slices were dissolved in 2% periodic acid (13). Radioactivity was determined in ACS cocktail (Amersham/Searle) in a Beckman LS-230 scintillation counter.Mrs were determined by sedimentation equilibrium with the meniscus depletion technique (14). The interference photographs were measured with an automatic scanner (15), and the data were evaluated by a computer program (16). Mrs were obtained from the in...

In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.