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

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

Automated LC-MS analysis and data extraction for high-throughput chemistry

High-throughput experimentation for chemistry and chemical biology has emerged as a highly impactful technology, particularly when applied to Direct-to-Biology. Analysis of the rich datasets which come from this mode of experimentation continues to be the rate-limiting step to reaction optimisation and the submission of compounds for biological assay. We present PyParse, an automated, accurate and accessible program for data extraction from high-throughput chemistry and provide real-life examples of situations in which PyParse can provide dramatic improvements in the speed and accuracy of analysing plate data. This software package has been made available through GitHub repository under an open-source Apache 2.0 licence, to facilitate the widespread adoption of high-throughput chemistry and enable the creation of standardised chemistry datasets for reaction prediction

Complexity, Development, and Evolution in Morphogenetic Collective Systems

Many living and non-living complex systems can be modeled and understood as collective systems made of heterogeneous components that self-organize and generate nontrivial morphological structures and behaviors. This chapter presents a brief overview of our recent effort that investigated various aspects of such morphogenetic collective systems. We first propose a theoretical classification scheme that distinguishes four complexity levels of morphogenetic collective systems based on the nature of their components and interactions. We conducted a series of computational experiments using a self-propelled particle swarm model to investigate the effects of (1) heterogeneity of components, (2) differentiation/re-differentiation of components, and (3) local information sharing among components, on the self-organization of a collective system. Results showed that (a) heterogeneity of components had a strong impact on the system's structure and behavior, (b) dynamic differentiation/re-differentiation of components and local information sharing helped the system maintain spatially adjacent, coherent organization, (c) dynamic differentiation/re-differentiation contributed to the development of more diverse structures and behaviors, and (d) stochastic re-differentiation of components naturally realized a self-repair capability of self-organizing morphologies. We also explored evolutionary methods to design novel self-organizing patterns, using interactive evolutionary computation and spontaneous evolution within an artificial ecosystem. These self-organizing patterns were found to be remarkably robust against dimensional changes from 2D to 3D, although evolution worked efficiently only in 2D settings.Comment: 13 pages, 8 figures, 1 table; submitted to "Evolution, Development, and Complexity: Multiscale Models in Complex Adaptive Systems" (Springer Proceedings in Complexity Series

PhotoAffinity bits : 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, however, it remains challenging to develop techniques that are both sufficiently high-throughput and sensitive. We present a fragment screening platform, termed PhABits (PhotoAffinity Bits), which utilises a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. We envision that the PhABits will be widely applicable to novel protein targets, identifying starting points in the development of therapeutic

Profiling sulfur(VI) fluorides as reactive functionalities for chemical biology tools and expansion of the ligandable proteome

Here, we report a comprehensive profiling study of sulfur(VI) fluorides (SVI-F) in the context of multiple chemical biology applications and illustrate that these motifs present an exciting opportunity to develop tools for a wide scope of protein targets. SVI-Fs are reactive functionalities that offer utility for targeting almost any protein, as they can modify multiple residues including Lys, Tyr, His and Ser. A panel of SVI-F functionalities were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acids. Subsequently, the reactivity of SVI-Fs with CAII and kinase proteins was investigated, in the context of both fragment binders and optimized probes. Finally, the performance of the SVI-F panel in chemoproteomic workflows was analyzed. The studies provided an in-depth understanding of the hydrolytic stability, protein reactivity and chemoproteomic utility of SVI-F functionalities that are suitable for direct incorporation into chemical tools. Such insights offer a valuable guide for the prospective design of SVI-F-containing ligands for various chemical biology workflows and demonstrate the wide range of proteins that SVI-Fs can capture, thus highlighting the opportunity for SVI-Fs to expand the liganded proteome

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

Selectively targeting the kinome-conserved lysine of PI3K (delta) 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 an alternative general strategy, to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors

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