A parallel selection against a column lacking protein was done to allow for the identification of molecules enriched by nontarget affinity. After three rounds of selection, the DNA tags of the eluted population were PCR amplified and sequenced, and the sequences were translated to identify structures of putative BCATm binders. Open in a separate window Scheme 2 Off-DNA Synthesis of Representative Compounds Selected by BCATmReagents and conditions: (a) diisopropyl azodicarboxylate, PPh3, tert-butyl 3-hydroxypyrrolidine-1-carboxylate; (b) (i) NaOH, THF-MeOH, H2O; (ii) HATU, DIEA, MeNH2HCl, AcCN; Rabbit Polyclonal to Chk2 (phospho-Thr383) (c) boronic acids, Pd(PPh3)4, Cs2CO3, THF/H2O, N2, 80 C, 2 h; (d) (i) 10% TFA/DCM; (ii) HATU, DIEA, R2COOH, AcCN; (e) H2 balloon, 10% Pd/C, MeOH; (f) (i) 10% TFA/DCM; (ii) HATU, DIEA, 5-bromothiophene-2-carboxylic acid, AcCN. Further analysis of the affinity selection output AZD3988 could be conveniently performed in a Spotfire cube view. in most tissues, BCATc is largely restricted to the central nervous system.1 BCAAs are essential amino acids that must be consumed in the diet AZD3988 of mammals since they do not possess the necessary enzymes to synthesize these molecules de novo. An increasing body of evidence supports the notion that BCAAs stimulate protein synthesis in skeletal muscle, with leucine being AZD3988 primarily responsible for the stimulation of protein synthesis after intake of a mixed meal.3 Conversely, it is also known that the product of leucine transamination, -keto isocaproic acid, inhibits protein degradation resulting in an overall accumulation of protein.4 A recent mouse BCATm knockout study demonstrated that lack of this enzyme increased the mices energy expenditure, associated with a futile protein turnover cycle, and protected them from obesity when subjected to a high fat diet.5 Furthermore, metabolomic profiling of obese versus lean humans reveals a BCAA-related metabolite signature that is suggestive of increased catabolism of BCAAs and correlated with insulin resistance.6 To further validate BCATm as a therapeutic target for the intervention of metabolic diseases, a suitable small molecular inhibitor is highly desirable. To date, no human BCATm inhibitors have ever been reported. Herein we report our effort in identifying small molecule inhibitors of human BCATm via DNA encoded library technology. More than 14 billion compounds from a superpool of various DNA encoded libraries of distinct scaffolds were screened against BCATm followed by off-DNA hit synthesis and activity confirmation. As a consequence, several potent BCATm chemotypes were discovered. In this report we describe the design and synthesis of a 34. 7-million-member DNA-encoded library and BCATm hits identified from this library. Encoded Library Technology (ELT) has proven to be an important lead discovery platform for various therapeutic targets.7?16 To increase library diversity and expand drug-like chemical space, we recently developed a trifunctional scaffold based novel DNA encoded library dubbed the CIA (carboxylic acid, iodide, and amine) library. Each of the trifunctional scaffolds contains a carboxylic acid, an aryl iodide group, and an Fmoc-protected amine group. Similar to our previously reported libraries, this library was synthesized using the split-and-pool strategy, beginning from a short sequence of duplex DNA stabilized by a synthetic hairpin (the headpiece).10,11 The design of the headpiece allows synthesis of the small molecule library starting from the free amine group, while DNA tags encoding the building blocks (BBs) are enzymatically ligated at the nonlinked end of the DNA duplex. In this instance, 44 CIA scaffolds (referred to as cycle 1 building blocks or BB1) were first installed onto the headpiece DNA starting material (1, Scheme 1) through amide bond formation followed by pooling and Fmoc-deprotection.11 After splitting into separate reaction wells, 265 cycle 2 boronic acids/ester building blocks (BB2) were introduced through on-DNA SuzukiCMiyaura cross-coupling reactions.17 This is our first reported application of on-DNA cross-coupling reactions in a library synthesis setting. After standard pooling, purification via spin filtration, and splitting into different reaction wells, 2976 unique cycle 3 building blocks (BB3) including carboxylic acids, sulfonyl chlorides, aldehydes, isocyanates, and heteroaryl chlorides were reacted with the amine group thus providing the final library with a size of 34.7 million. After pooling and purification, the DNA sequence was then closed with a final tag that encodes a library identifier, a priming region for PCR amplification, and a degenerate sequence to control for PCR amplification biases. Open in a separate window Scheme 1 Synthesis of the Three Cycle DNA Encoded CIA Library The affinity selections were performed on a chemically biotinylated human BCATm protein construct. The target protein was immobilized on a streptavidin agarose resin packed AZD3988 column. After immobilization, the column was washed with buffer containing 1 mM biotin to ensure occupation of all biotin binding sites. The immobilized protein was then exposed to the on-DNA library prior to extensive washing to remove nonbinders. Bound library molecules were eluted by heat denaturation of the protein at 80 C. The eluant was then incubated with fresh immobilized protein to start a new round of affinity selection. A parallel selection against a column lacking protein was done.