As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)-pyrrolidin-3-yl)oxy)-N-methyl-2′-(methylsulfonamido)-[1,1′-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki−Miyaura cross-coupling showed BCATm inhibitory activity with IC 50 = 2.0 μM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors.

The requirement for TRPV6 for vitamin D-dependent intestinal calcium absorption in vivo has been examined by using vitamin D-deficient TRPV6 null mice and littermate wild-type mice. Each of the vitamin D-deficient animals received each day for 4 days 50 ng of 1,25-dihydroyvitamin D 3 in 0.1 ml of 95% propylene glycol:5% ethanol vehicle or vehicle only. Both the wild-type and TRPV6 null mice responded equally well to 1,25-dihydroxyvitamin D 3 in increasing intestinal calcium absorption. These results, along with our microarray data, demonstrate that TRPV6 is not required for vitamin D-induced intestinal calcium absorption and may not carry out a significant role in this process. These and previous results using calbindin D9k null mutant mice illustrate that molecular events in the intestinal calcium absorption process in response to the active form of vitamin D remain to be defined.A primary function of vitamin D is to markedly increase intestinal absorption of calcium and phosphate (1). During the 1950s, this absorption was shown to be primarily an active calcium transport process and an independent active phosphate transport process (2, 3). With the discovery of the vitamin D endocrine system came the understanding that this process was directly stimulated by the hormonal form of vitamin D, 1␣,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) (4). Because this hormonal form of vitamin D is regulated in response to the need for calcium, it became clear that the endogenous factor discovered by Nicolaysen and Egg-Larsen is the vitamin D endocrine system producing the final vitamin D hormone, 1,25-(OH) 2 D 3 (4, 5). There is still debate whether vitamin D further influences the diffusional component of calcium absorption, taking place at high intestinal levels of calcium (6, 7).The molecular mechanism underlying active calcium transport in response to vitamin D began unraveling with the discovery of calbindin D 9k by Wasserman and colleagues (8). Schachter and colleagues (9) also found a transporter responsive to vitamin D. Others have reported that vitamin D stimulates the basal lateral membrane calcium ATPase believed to be a calcium transporter (10, 11). These components have been put together in a diagrammatic fashion to present the current hypothesis of how 1,25-(OH) 2 D 3 stimulates active intestinal calcium absorption. TRPV6 is a calcium channel protein clearly induced by 1,25-(OH) 2 D 3 (12). Calcium entering through this channel is believed to associate with calbindin D 9k , which serves as a shuttle for calcium, presenting it to the basal lateral membrane calcium ATPase. This step provides the energy input for the transfer of calcium against a concentration gradient. All three of these genes are clearly under the influence of the active form of vitamin D (13). Unfortunately not all evidence is currently in support of this hypothesis. Transgenic mice in which calbindin D 9k has been eliminated have shown that calbindin D 9k is not required for 1,25-(OH) 2 D 3 -stimulated intestinal calcium absorption (14, 15). Fo...

The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.

To identify BCATm inhibitors suitable for in vivo study, Encoded Library Technology (ELT) was used to affinity screen a 117 million member benzimidazole based DNA encoded library, which identified an inhibitor series with both biochemical and cellular activities. Subsequent SAR studies led to the discovery of a highly potent and selective compound, 1-(3-(5-bromothiophene-2-carboxamido)cyclohexyl)-N-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole-5-carboxamide (8b) with much improved PK properties. X-ray structure revealed that 8b binds to the active site of BACTm in a unique mode via multiple H-bond and van der Waals interactions. After oral administration, 8b raised mouse blood levels of all three branched chain amino acids as a consequence of BCATm inhibition.

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The original version of this Article omitted the following from the Acknowledgements: 'We thank Robert Kirkpatrick for implementing the high throughput protein design strategy that enabled screening and triage of essential A. baumannii targets, based on whole genome sequencing and annotation of BM4454 strain; and Stephanie Van Horn, Allan Kwan, Elizabeth Valoret for A. baumannii genome sequencing and annotation.' Also, the original version omitted an acknowledgement to Prof. Lydia Tabernero as one of our collaborators for supplying the purified proteins used in the Tuberculosis screen. This has been corrected in both the PDF and HTML versions of the Article.