Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 µL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

Long-Acting Human Growth Hormone Analogue by Noncovalent Albumin Binding.

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.