| Project Detail |
A fundamental feature of biology is that when evolution happens upon a new function, both the genotype and phenotype need to be amplifiable. In other words DNA must be able to copy itself, while also leading to the creation of many copies of its encoded protein. This basic precept of encoded systems in biology has been applied across the more biological encoded library systems (phage display, mRNA display, SELEX), but has never been applied with small molecule DNA encoded libraries (DELs). We propose here that amplification of molecular or functional signals in DNA encoded library (DEL) technology is the next logical evolution of the field. As a proof-of-concept for how our work will open new areas of investigation for encoded libraries we will show a first-in-class method for how DELs could tackle one of the currently most pressing challenges in drug discovery: the identification of small molecules that degrade target proteins.Screening compound libraries for binding or inhibitory activity against target proteins is an essential part of early drug discovery. High-throughput screening (HTS) facilities, however, come with a large infrastructural burden. DNA encoded libraries (DELs) offer a radically different approach to initial hit-identification. DEL technology uses DNA tags to track the synthetic history of individual members in a split-and-pool combinatorial synthesis scheme. Since each compound’s identity is encoded in its covalently connected DNA, compounds can be pooled and then selected against target proteins in an affinity selection. After selection, the retained molecules are washed from beads and their DNA tags are sequenced with next generation sequencing (NGS). DNA sequencing is the final data readout, and the hope is that NGS read-counts give a somewhat faithful representation of binding affinity. Unfortunately, this elegant concept only works well for extremely potent binders - with moderate binders, data quality in DEL selections is often poor. Furthermore, functional selections (i.e. where a specific function rather than simple binding is desired) are almost absent from pooled DEL selections despite the fact that for classical screening methods functional selections are by far the most common (for example, enzyme inhibition, GPCR signal transduction). In simple terms we look at it like this: when a small molecule linked to a DNA interacts with a target, we need to amplify that signal or couple it to a functional read-out if we hope for the field to advance.So, starting from the overarching concept that DEL selections need to amplify their molecular signal we have conceived of three ways in which this could happen. We believe there are likely many other ways to make this happen, and we believe our results will stimulate others in the field to pursue the concept. The projects are:1.When a protein binds to a DEL, could we initiate the transcription of RNA copies of the DNA tag? In this way the signal would be amplified and the final step would be simple isolation and sequencing of RNA (i.e. RNAseq) - an extremely optimized technique used in nearly every biology and chemical biology lab in the world, including our own.2.Terminyl deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3’ ends of single-stranded or duplex DNA. If we express our target proteins as fusions with TdT, could DEL members that bind to the target protein be extended at their 3’ ends? If so the DNA tag of DEL members bound to the target protein would grow in length, giving a signal amplification with the following logic: longer DNA tag = better binder.3.Could DEL selections select for function rather than binding? We will use the example of Ubiquitin-transfer to demonstrate how DELs can be selected for a catalytic transfer event. This project will extend the reach of DEL technology to the extremely challenging problem of identifying small molecules that can trigger protein degradation.The approaches outlined would amplify the binding or functional signal, while simultaneously eliminating the need for capricious affinity selections. Not only will these innovations change current practice, but they will open new doors for DELs as in the case of Project 3, where small molecules will be selected that degrade the proteins they bind with. |
