UC Irvine

The Human Genome Project ultimately aimed to translate DNA sequence into drugs. With the draft in hand, the Molecular Libraries Program set out to prosecute all genome-encoded proteins for drug discovery with automated high-throughput screening (HTS). This ambitious vision remains unfulfilled, even while innovations in sequencing technology have fully democratized access to genome-scale sequencing. Why? While the central dogma of biology allows us to chart the entirety of cellular metabolism through sequencing, there is no direct coding for chemistry. The rules of base pairing that relate DNA gene to RNA transcript and amino acid sequence do not exist for relating small molecule structure with macromolecular binding partner and subsequently cellular function. Obtaining such relationships genome-wide is unapproachable via state-of-the-art HTS, akin to attempting genome-wide association studies using turn-of-the-millennium Sanger DNA sequencing.

Our laboratory has been engaged in a multi-pronged technology development campaign to revolutionize molecular screening through miniaturization in pursuit of genome-scale drug discovery capabilities. We employed DNA-encoded library (DEL) synthesis principles in the development of solid-phase DELs prepared on microscopic beads, each harboring 100 fmol of a single library member and a DNA tag whose sequence describes the structure of the library member. Loading these DEL beads into 100-pL microfluidic droplets followed by on-line photocleavage, incubation, fluorescence-activated droplet sorting, and DNA sequencing of the sorted DEL beads reveals the chemical structures of bioactive compounds. This scalable library synthesis and screening platform has proven useful in several proof-of-concept projects involving current clinical targets. Moving forward, we face the problem of druggability and proteome-scale assay development. Developing biochemical or cellular assays for all genome-encoded targets is not scalable and likely impossible as most proteins have ill-defined or unknown activity and may not function outside of their native context.

In this dissertation I describe the utility of selective translation modulation as a modality that circumvents canonical druggability constraints while also raising the prospect of in vitro transcription translation (IVTT) as a universal biochemical assay for screening. In Chapter 2, I present the development of an IVTT activity assay by fusing a GFP reporter to various gene sequences that are known to be selectively inhibited by compound PF846. Wheat germ IVTT was miniaturized to microfluidic droplets and demonstrated to exhibit excellent assay quality both in response to positive control PF846 and beads displaying a photocleavable broad-spectrum translation inhibitor, hygromycin B. Chapter 3 describes the screening of a 5,348-member DEL of custom translation inhibitor analogs. Screening a proof-of-concept PCSK9-GFP reporter yielded several hits that selectively inhibited PCSK9-GFP IVTT. Chapter 4 details the screening of an unbiased 55,296-member DEL to discover novel translation inhibitor chemotypes. These studies set the stage for scaling microfluidic IVTT DEL screening to the human proteome, irrespective of mature protein function, to fulfill the Genome Project's vision of proteome-wide control over cellular pharmacology.