Applications of Next Generation Sequencing in the Field of Chemical Biology

Chemical biology is the study of biological systems accelerated primarily by chemical methods and the assistance of technologies from biology. Powerful techniques such as Next Generation Sequencing (NGS) made huge impacts on this field and paved the way for new technologies such as DNA encoded libraries that dramatically accelerated the discovery of new protein ligands. We present here two NGS applications in chemical biology that expand and offer a new viewpoint on existing ideas. DNA alkylating drugs such as temozolomide (TMZ) or streptozotocin (STZ) are known to be electrophiles that modify DNA and cause the cell death of fast replicating cells. Their impact on RNA has been studied less thoroughly, as RNA is typically regarded as being short-lived. We adapted sequencing techniques used to probe secondary and tertiary structures of RNA to measure the damage caused by alkylating drugs. With this approach, we have shown that TMZ and STZ alkylate RNA selectively at G in a non-sequence dependent manner. We have validated our approach by comparing these treatments to the effect of the electrophilic small molecule dimethylsulfate (DMS). Furthermore, we have measured for the first time the alkylation profile of trimethylsilyl diazomethane by RNA sequencing and showed that it reacts similarly to DMS, both in vitro and in vivo. We believe that this method is widely applicable and can be used to study the alkylation patterns of more complex natural product. We applied the knowledge of NGS and bioinformatics and transferred it to DNA encoded libraries (DELs), culminating in a new open-source toolkit written in python. This toolkit enabled and accelerated our studies of DELs by offering a convenient command-line interface to create these combinatorial libraries in situ. Furthermore, the toolkit assists in evaluating the NGS results by offering easy-to-use tools and helps to understand the results by generating quick standard reports. Finally, we created a DNA encoded library where we diversified a DNA-bound azide with a library of alkynes by a copper(I)-catalysed cycloaddition, which we subsequently encoded by splint-ligation. We used the same azide to generate a second sublibrary by reducing it to an amine and diversifying it with carboxylic acids. We encoded for these acids using the same codon space. Then, we introduced a unique identifier for each sublibrary encoding for the reaction type used, a concept we termed reaction encoding. This approach increases the diversity of one point by utilising several small sets of differently functionalised building blocks, making the purchase of large building block libraries unnecessary. Furthermore, it allows analysing structure-activity relationships not only across building blocks but also across connectivity types

The pKa of Bronsted acids controls their reactivity with diazo compounds

We study the O-alkylation of phosphate groups by alkyl diazo compounds in a range of small molecules and biopolymers. We show that the relatively high pKa of phosphate in comparison to the other naturally occurring Brønsted acids can be exploited to control alkylation selectivity. We provide a simple protocol for chemical modification of some of the most important instances of phosphates in natural compounds including in small molecule metabolites, nucleic acids, and peptides

Profiling the Nucleobase and Structure Selectivity of Anticancer Drugs and other DNA Alkylating Agents by RNA Sequencing

Drugs that covalently modify DNA are components of most chemotherapy regimens, often serving as first‐line treatments. Classically, the reactivity and selectivity of DNA alkylating agents has been determined in vitro with short oligonucleotides. A statistically sound analysis of sequence preferences of alkylating agents is untenable with serial analysis methods because of the combinatorial explosion of sequence possibilities. Next‐generation sequencing (NGS) is ideally suited for the broad characterization of sequence or structure selectivities because it analyzes many sequences at once. Herein, NGS is used to report on the chemoselectivity of alkylating agents on RNA and this technology is applied to the previously uncharacterized alkylating agent trimethylsilyl diazomethane

A DNA-Encoded Chemical Library Incorporating Elements of Natural Macrocycles

Abstract Here we show a seven-step chemical synthesis of a DNA-encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide-polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of the custom synthesis of bifunctional building block libraries. This study outlines the importance of careful retrosynthetic design in DNA-encoded libraries, while revealing areas where new DNA synthetic methods are needed

A new water soluble copper N-heterocyclic carbene complex delivers mild O6G-selective RNA alkylation

We show here that copper carbenes generated from diazo acetamides alkylate single RNAs, mRNAs, or pools of total transcriptome RNA, delivering exclusively alkylation at the O 6 position in guanine (O 6 G). Although the reaction is effective with free copper some RNA fragmentation occurs, a problem we resolve by developing a novel water-stable copper N-heterocyclic carbene complex. Carboxymethyl adducts at O 6 G are known mutagenic lesions in DNA but their relevance in RNA biochemistry is unknown. As a case-in-point we re-examine an old controversy regarding whether O 6 G damage in RNA is susceptible to direct RNA repair

DNA Damaging Agents in Chemical Biology and Cancer

Despite their toxicity, DNA alkylating drugs remain a cornerstone of anticancer therapy. The classical thinking was that rapidly dividing tumour cells left more of its DNA in an exposed single-stranded state, making these rapidly dividing cells more susceptible to alkylating drugs. As our understanding of DNA repair pathways has matured it is becoming clear that compromised DNA repair – a hallmark of cancer – plays a role as well in defining the therapeutic window of these toxic drugs. Hence, although new alkylating motifs are unlikely to progress through the clinic, the legacy of these medicines is that we now understand the therapeutic potential of targeting DNA damage repair pathways. Here we look at the history of alkylating agents as anticancer drugs, while also summarizing the different mechanistic approaches to covalent DNA modification. We also provide several case studies on how insights into compromised DNA repair pathways are paving the way for potent and less toxic targeted medicines against the DNA damage response

An assessment of the mutational load caused by various reactions used in DNA encoded libraries

DNA encoded libraries have become an essential hit-finding tool in early drug discovery. Recent advances in synthetic methods for DNA encoded libraries have expanded the available chemical space, but precisely how each type of chemistry affects the DNA is unstudied. Available assays to quantify the damage are limited to write efficiency, where the ability to ligate DNA onto a working encoded library strand is measured, or qPCR is performed to measure the amplifiability of the DNA. These measures read signal quantity and overall integrity, but do not report on specific damages in the encoded information. Herein, we use next generation sequencing (NGS) to measure the quality of the read signal in order to quantify the truthfulness of the retrieved information. We identify CuAAC to be the worst offender in terms of DNA damage amongst commonly used reactions in DELs, causing an increase of G → T transversions. Furthermore, we show that the analysis provides useful information even in fully elaborated DELs; indeed we see that vestiges of the synthetic history, both chemical and biochemical, are written into the mutational spectra of NGS datasets

A method to identify small molecule/protein pairs susceptible to protein ubiquitination

Although using DNA encoded libraries (DELs) to find small molecule binders of target proteins is well-established, identifying DEL hits for functions other than binding remains challenging. We demonstrate here a technique where pools of DNA-linked small molecules are mixed with pools of DNA-linked protein targets and optimal small/protein pairs are identified based on their ability to catalyze the transfer of ubiquitin (Ub) onto the target proteins. Since the transfer of Ub is the first step in the tagging of proteins for proteasomal destruction, finding small molecules that can selectively reprogram Ub-transfer is one of the great challenges in contemporary drug development. Our work provides the framework for a new type of functional DEL screen that matches small molecule Ub-transfer catalysts with their optimal protein substrates. We believe the technology could be especially useful in discovering and optimizing molecular glue degraders

A DNA-Encoded Macrocycle Library that Resembles Natural Macrocycles

Herein we perform a seven-step chemical synthesis of a DNA-encoded macrocycle library (DEML) on DNA. Inspired by polyketide and mixed peptide-polyketide natural products, the library was designed to incorporate rich backbone diversity. Achieving this diversity, however, comes at the cost of custom synthesis of bifunctional building block libraries. Our work outlines the importance of careful retrosynthetic design in DNA-encoded libraries, while revealing areas where new DNA synthetic methods are needed. </div