Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, ...histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.
mRNA display is a powerful technology to screen libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands. These cyclic peptides are ...particularly well suited to challenging protein targets that have been difficult to drug with small molecules. However, target choice can still be limited as screens are typically performed against purified proteins which often demands the use of isolated domains and precludes the use of aggregation‐prone targets. Here, we report a method to perform mRNA display selections in mammalian cell lysates without the need for prior target purification, vastly expanding the potential target scope of mRNA display. We have applied the methodology to identify low to sub‐nanomolar peptide binders for two targets: a NanoLuc subunit (LgBiT) and full‐length bromodomain‐containing protein 3 (BRD3). Our cyclic peptides for BRD3 were found to bind to the extraterminal (ET) domain of BRD3 and the closely related BRD proteins, BRD2 and BRD4. While many chemical probes exist for the bromodomains of BRD proteins, the ET domain is relatively underexplored, making these peptides valuable additions to the BRD toolbox.
An important consideration in drug discovery is the prioritization of tractable protein targets that are not only amenable to binding small molecules, but also alter disease biology in response to ...small molecule binding. Covalent fragment-based drug discovery has emerged as a powerful approach to aid in the identification of such protein targets. The application of irreversible binding mechanisms enables the identification of fragment hits for challenging-to-target proteins, allows proteome-wide screening in a cellular context, and makes it possible to determine functional effects with modestly potent ligands without the requirement for extensive compound optimization. Here, we provide an overview of recent approaches to covalent fragment-based screening and discuss how these have been applied to establish the tractability of unexplored binding sites on protein targets.
Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is ...limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.
There are an estimated 200,000 to 400,000 cases of visceral leishmaniasis (VL) annually. A variety of factors are taken into account when considering the best therapeutic options to cure a patient ...and reduce the risk of resistance, including geographical area, malnourishment and HIV coinfection. Pooled analyses combine data from many studies to answer specific scientific questions that cannot be answered with individual studies alone. However, the heterogeneity of study design, data collection, and analysis often makes direct comparison difficult. Individual Participant Data (IPD) files can be standardised and analysed, allowing detailed analysis of this merged larger pool, but only a small fraction of systematic reviews and meta-analyses currently employ pooled analysis of IPD. We conducted a systematic literature review to identify published studies and studies reported in clinical trial registries to assess the feasibility of developing a VL data sharing platform to facilitate an IPD-based analysis of clinical trial data. Studies conducted between 1983 to 2015 that reported treatment outcome were eligible.
From the 2,271 documents screened, 145 published VL clinical trials were identified, with data from 26,986 patients. Methodologies varied for diagnosis and treatment outcomes, but overall the volume of data potentially available on different drugs and dose regimens identified hundreds or possibly thousands of patients per arm suitable for IPD pooled meta-analyses.
A VL data sharing platform would provide an opportunity to maximise scientific use of available data to enable assessment of treatment efficacy, contribute to evidence-based clinical management and guide optimal prospective data collection.
Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, ...we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn
2+
binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.
A photoreactive fragment screening platform employing direct-to-biology high-throughput chemistry (D2B-HTC) for the rapid iterative synthesis and screening of libraries of photoaffinity bits.
Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a ...disease‐modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment‐screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment–protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.
PhotoAffinity Bit (PhABit) is a photoreactive fragment‐screening platform to covalently capture fragment–protein interactions. Hits can be profiled and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is widely applicable to novel protein targets, identifying starting points in the development of therapeutics.
The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential ...therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probe‐based competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest.
Selectivity snapshots with PALs: Photoaffinity probes were designed to target the cyclin‐dependent kinase family, and found to competitively enrich CDKs from cell lysates. Subsequently, a biochemical photoaffinity displacement assay was developed to measure compound potency.
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