Photoaffinity labelling displacement assay using multiple recombinant protein domains.

The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR-FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.

mRNA Display in Cell Lysates Enables Identification of Cyclic Peptides Targeting the BRD3 Extraterminal Domain.

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.

A Chemo- and Regioselective Tandem [3 + 2]Heteroannulation Strategy for Carbazole Synthesis: Combining Two Mechanistically Distinct Bond-Forming Processes.

A modular approach to prepare tri- and tetracyclic carbazoles by a sequential [3 + 2]heteroannulation is described. First, optimization of Pd-catalyzed Buchwald-Hartwig amination followed by C/N-arylation in a one-pot process is established. Second, mechanistic analyses identified the origins of chemo- and regioselective sequential control of both bond-forming steps. Finally, the substrate scope is demonstrated by the preparation of a range of tri- and tetracyclic carbazoles, including expedient access to several natural products and anti-cancer agents.

One-Step Synthesis of Photoaffinity Probes for Live-Cell MS-Based Proteomics.

We present a one-step Ugi reaction protocol for the expedient synthesis of photoaffinity probes for live-cell MS-based proteomics. The reaction couples an amine affinity function with commonly used photoreactive groups, and a variety of handle functionalities. Using this technology, a series of pan-BET (BET: bromodomain and extra-terminal domain) selective bromodomain photoaffinity probes were obtained by parallel synthesis. Studies on the effects of photoreactive group, linker length and irradiation wavelength on photocrosslinking efficiency provide valuable insights into photoaffinity probe design. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles.

Quantitative MS-Based Proteomics: Comparing the MCF-7 Cellular Response to Hypoxia and a 2-Oxoglutarate Analogue.

The hypoxia-inducible factors (HIFs) are key transcription factors in determining cellular responses involving alterations in protein levels in response to limited oxygen availability in animal cells. 2-Oxoglutarate-dependent oxygenases play key roles in regulating levels of HIF and its transcriptional activity. We describe MS-based proteomics studies in which we compared the results of subjecting human breast cancer MCF-7 cells to hypoxia or treating them with a cell-penetrating derivative (dimethyl N-oxalylglycine; DMOG) of the stable 2OG analogue N-oxalylglycine. The proteomic results are consistent with reported transcriptomic analyses and support the proposed key roles of 2OG-dependent HIF prolyl- and asparaginyl-hydroxylases in the hypoxic response. Differences between the data sets for hypoxia and DMOG might reflect context-dependent effects or HIF-independent effects of DMOG.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

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.

A Photoaffinity Displacement Assay and Probes to Study the Cyclin-Dependent Kinase Family.

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.

Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition.

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.

Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases.

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual "ping-pong" mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.

Covalent fragment-based drug discovery for target tractability.

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.

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry.

The screening of covalent or 'reactive' fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

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.

Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments.

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors.

Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.

Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1)WT or BRD4(1)L94C, to determine their selectivity for BRD4(1)L94C over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1)L94C over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

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 Zn2+ 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.

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

A Turing Test for Molecular Generators.

Machine learning approaches promise to accelerate and improve success rates in medicinal chemistry programs by more effectively leveraging available data to guide a molecular design. A key step of an automated computational design algorithm is molecule generation, where the machine is required to design high-quality, drug-like molecules within the appropriate chemical space. Many algorithms have been proposed for molecular generation; however, a challenge is how to assess the validity of the resulting molecules. Here, we report three Turing-inspired tests designed to evaluate the performance of molecular generators. Profound differences were observed between the performance of molecule generators in these tests, highlighting the importance of selection of the appropriate design algorithms for specific circumstances. One molecule generator, based on match molecular pairs, performed excellently against all tests and thus provides a valuable component for machine-driven medicinal chemistry design workflows.

Small-molecules that covalently react with a human prolyl hydroxylase - towards activity modulation and substrate capture.

We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. Reversible active site binding of electrophile bearing compounds enables susbsequent covalent reaction with a lysine residue (K408) in the flexible C-terminal region of PHD2 to give a modified protein that retains catalytic activity.

Systematic review of clinical trials assessing the therapeutic efficacy of visceral leishmaniasis treatments: A first step to assess the feasibility of establishing an individual patient data sharing platform.

BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure-activity relationship studies leading to the discovery of an n-octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 µM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n-octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n-octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell-permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.