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Peroxide-cleavable arylboronates are stable linkers for the generation of efficacious antibody–drug conjugates.

SCFSkp2/Cks1is an E3 ubiquitin ligase, whose substrate specificity is determined by the oncogenic F-box protein Skp2 and the adaptor protein Cks1. A principal target of SCFSkp2/Cks1is the cyclin-dependent kinase inhibitor p27. Elevated levels of Skp2 and reduced levels of p27 are common in a variety of cancers, and there is consequently a need to develop effective inhibitors of the Skp2-p27 interaction. However, conventional small-molecule approaches are challenging due to the extended bi-molecular interface that spans both Skp2 and Cks1, the lack of suitable binding pockets on this surface, and the intrinsically disordered nature of p27. Here, we develop macrocyclic peptides capable of binding to SCFSkp2/Cks1with nanomolar affinities, an enhancement of almost two orders of magnitude over the natural p27 peptide. We show that these macrocyclic peptides inhibit p27 ubiquitination in vitro, restore p27 levels in a breast cancer cell line, and reduce cell proliferation.

The development of divinylpyrimidine (DVP) reagents for the synthesis of antibody–drug conjugates (ADCs) with in vivo efficacy and tolerability is reported.

CK2 is a ubiquitous protein kinase, with key roles in the regulation of cell growth and proliferation. In particular, CK2 acts as an anti-apoptotic protein and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is to inhibit the formation of the holoenzyme complex. This is possible, with the most notable example being CAM7117. However, CAM7117 contains unnatural amino acids, residues not directly involved in the binding to CK2, and its size limits further optimisations. In this work, an iterative cycle of enzymatic assays, X-ray crystallography, molecular modelling and cellular assays were used to develop a functionalisable chemical probe for the CK2α/β PPI. The lead peptide, P8C9, successfully binds to CK2α at the protein-protein interaction site, is easily synthesisable and functionalisable, highly stable in serum and small enough to accommodate further optimisation. Furthermore, its cell-permeable analogues, TAT-P8C9 and R3-P8C9, successfully inhibit cell proliferation. TAT-P8C9 and R3-P8C9 can serve as true chemical probes to further understand the intracellular pathways involving CK2, as well as aiding the development of novel CK2 PPI inhibitors for therapeutic use.

The protein kinase Aurora A, and its close relative, Aurora B, regulate human cell division. Aurora A is frequently overexpressed in cancers of the breast, ovary, pancreas and blood, provoking genome instability and resistance to anti-mitotic chemotherapy. Intracellular localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. Here, we have used fragment-based, structure-guided lead discovery to develop small-molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). These compounds act by novel mechanism compared to existing Aurora A inhibitors and they are highly specific to Aurora A over Aurora B. We identify a biophysically, structurally and phenotypically validated lead compound,CAM2602, exhibits oral bioavailability, favourable pharmacokinetics, pharmacodynamic biomarker modulation, and arrest of growth in tumour xenografts. Consistent with our original finding that Aurora A overexpression drives taxane-resistance in cancer cells,CAM2602synergizes with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a structural and chemical blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

Tuberculosis treatment requires months-long combination chemotherapy with multiple drugs, with shorter treatments leading to relapses. A major impediment to shortening treatment is that Mycobacterium tuberculosis becomes tolerant to the administered drugs, starting early after infection and within days of infecting macrophages. Multiple lines of evidence suggest that macrophage-induced drug tolerance is mediated by mycobacterial drug efflux pumps. Here, using assays to directly measure drug efflux, we find that M. tuberculosis transports the first-line antitubercular drug rifampicin through a proton gradient-dependent mechanism. We show that verapamil, a known efflux pump inhibitor, which inhibits macrophage-induced rifampicin tolerance, also inhibits M.tuberculosis rifampicin efflux. As with macrophage-induced tolerance, the calcium channel-inhibiting property of verapamil is not required for its inhibition of rifampicin efflux. By testing verapamil analogs, we show that verapamil directly inhibits M. tuberculosis drug efflux pumps through its human P-glycoprotein (PGP)-like inhibitory activity. Screening commonly used drugs with incidental PGP inhibitory activity, we find many inhibit rifampicin efflux, including the proton pump inhibitors (PPIs) such as omeprazole. Like verapamil, the PPIs inhibit macrophage-induced rifampicin tolerance as well as intramacrophage growth, which has also been linked to mycobacterial efflux pump activity. Our assays provide a facile screening platform for M. tuberculosis efflux pump inhibitors that inhibit in vivo drug tolerance and growth.

Stapling is a macrocyclisation method that connects amino acid side chains of a peptide to improve its pharmacological properties. We describe an approach for stapled peptide preparation and biochemical evaluation that combines recombinant expression of fusion constructs of target peptides and cysteine-reactive divinyl-heteroaryl chemistry, as an alternative to solid-phase synthesis. We then employ this workflow to prepare and evaluate BRC-repeat-derived inhibitors of the RAD51 recombinase, showing that a diverse range of secondary structure elements in the BRC repeat can be stapled without compromising binding and function. Using X-ray crystallography, we elucidate the atomic-level features of the staple moieties. We then demonstrate that BRC-repeat-derived stapled peptides can disrupt RAD51 function in cells following ionising radiation treatment.

Herein, we present sp3-rich fragments, that display optimal physicochemical properties and exit vectors ideal for fragment-based lead discovery.

The caseinolytic protease complex ClpXP is an important house-keeping enzyme in prokaryotes charged with the removal and degradation of misfolded and aggregated proteins and performing regulatory proteolysis. Dysregulation of its function, particularly by inhibition or allosteric activation of the proteolytic core ClpP, has proven to be a promising strategy to reduce virulence and eradicate persistent bacterial infections. Here, we report a rational drug-design approach to identify macrocyclic peptides which increase proteolysis by ClpP. This work expands the understanding of ClpP dynamics and sheds light on the conformational control exerted by its binding partner, the chaperone ClpX, by means of a chemical approach. The identified macrocyclic peptide ligands may, in the future, serve as a starting point for the development of ClpP activators for antibacterial applications.

RAS proteins are central in the proliferation of many types of cancer, but a general approach toward the identification of pan-mutant RAS inhibitors has remained unresolved. In this work, we describe the application of a binding pharmacophore identified from analysis of known RAS binding peptides to the design of novel peptides. Using a chemically divergent approach, we generated a library of small stapled peptides from which we identified compounds with weak binding activity. Exploration of structure-activity relationships (SARs) and optimization of these early compounds led to low-micromolar binders of KRAS that block nucleotide exchange.

The caseinolytic protease complex ClpXP is an important house-keeping enzyme in prokaryotes charged with the removal and degradation of misfolded and aggregated proteins and performing regulatory proteolysis. Dysregulation of its function, particularly by inhibition or allosteric activation of the proteolytic core ClpP, has proven to be a promising strategy to reduce virulence and eradicate persistent bacterial infections. Here, we report a rational drug-design approach to identify macrocyclic peptides which allosterically increase proteolysis by ClpP. This work expands the understanding of ClpP dynamics and sheds light on the conformational control exerted by its binding partner, the chaperone ClpX, by means of a chemical approach. The identified macrocyclic peptide ligands may, in the future, serve as a starting point for the development of ClpP activators for antibacterial applications.

Leucine-rich repeat-containing G-protein receptor 5 (LGR5) has been characterised as a stem cell and cancer stem cell marker. Previous analyses of LGR5 transcript levels indicate high level expression discriminates malignancies such as colorectal cancer (CRC) and pre-B acute lymphoblastic leukaemia (pre-B ALL) from healthy tissues suggesting LGR5 protein expression may provide a molecular handle for prognosis and treatment.We have developed highly specific, high affinity antibodies to the extracellular domain of human LGR5 (α-LGR5) that detect high LGR5 protein levels in colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL. In contrast, there is low to undetectable levels of LGR5 protein in normal colon and rectal epithelia, liver, ovarian tissues, brain and immune cell types.LGR5 is rapidly internalised from the plasma membrane and trafficked to intracellular vesicular compartments including lysosomes. Treatment of high LGR5-expressing CRC and pre-B ALL cancer cell lines with an antibody-drug conjugate version of α-LGR5 (α-LGR5-ADC) lead to effective cell killing at nanomolar concentrations. Interventional treatment of pre-B ALL tumours with α-LGR5-ADC in vivo led to rapid tumour attrition. We further demonstrated the therapeutic utility of humanised α-LGR5 by using the corresponding scFv fragment for the generation of α-LGR5 chimeric antigen receptors (CARs) and a Bispecific T cell Engager (BiTE). α-LGR5-CAR-NK cells were effective at killing LGR5-expressing cells while α-LGR5/α-CD3 BiTEs induce T cell activation and killing of NALM6 cells by cytotoxic CD8+ T cells.Taken together, this study establishes α-LGR5-based therapeutic modalities that effectively discriminate and target CRC, HCC and pre-B ALL tumour cells.One Sentence SummaryWe generated novel antibodies against the cancer cell marker LGR5, validated diagnostic use in prioritizing specific cancer types for targeting, and developed antibody-based therapeutics.

Herein we report the development of a methodology for the dual-functionalisation of IgG antibodies. This is accomplished through the combination of disulfide rebridging divinylpyrimidine technology, with bicyclononyne and methylcyclopropene handles to facilitate sequential SPAAC and IEDDA reactions. Advantageously, the strategy does not require metal catalysis and avoids the need for purification between functionalisation steps.

Amino acid modification plays an important role across several fields, including synthetic organic chemistry, materials science, targeted drug delivery and the probing of biological function. Although a myriad of methods now exist for the modification of peptides or proteins, many of these target a handful of the most reactive proteinogenic amino acids. Photocatalysis has recently emerged as a mild approach for amino acid modification, generating a sizable toolbox of reactions capable of modifying almost all of the canonical amino acids. These reactions are characterised by their mild, physiologically compatible conditions, greatly enhancing their usefulness for amino acid modification. This review aims to introduce the field of photocatalytic amino acid modification and discusses the most recent advances.

Antibody-drug conjugates (ADCs) harness the highly specific targeting capabilities of an antibody to deliver a cytotoxic payload to specific cell types. They have garnered widespread interest in drug discovery, particularly in oncology, as discrimination between healthy and malignant tissues or cells can be achieved. Nine ADCs have received approval from the US Food and Drug Administration and more than 80 others are currently undergoing clinical investigations for a range of solid tumours and haematological malignancies. Extensive research over the past decade has highlighted the critical nature of the linkage strategy adopted to attach the payload to the antibody. Whilst early generation ADCs were primarily synthesised as heterogeneous mixtures, these were found to have sub-optimal pharmacokinetics, stability, tolerability and/or efficacy. Efforts have now shifted towards generating homogeneous constructs with precise drug loading and predetermined, controlled sites of attachment. Homogeneous ADCs have repeatedly demonstrated superior overall pharmacological profiles compared to their heterogeneous counterparts. A wide range of methods have been developed in the pursuit of homogeneity, comprising chemical or enzymatic methods or a combination thereof to afford precise modification of specific amino acid or sugar residues. In this review, we discuss advances in chemical and enzymatic methods for site-specific antibody modification that result in the generation of homogeneous ADCs.

CK2 is a protein kinase that plays important roles in many physio-pathological cellular processes. As such, the development of chemical probes for CK2 has received increasing attention in the past decade with more than 40 lead compounds developed. In this review, we aim to provide the reader with a comprehensive overview of the chemical probes acting outside the highly-conserved ATP-site developed to date. Such probes belong to different classes of molecules spanning from small molecules to peptides, act with a range of mechanisms of action and some of them present themselves as promising tools to investigate the biology of CK2 and therefore develop therapeutics for many disease areas including cancer and COVID-19.

CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.

Antibody-drug conjugates (ADCs) are valuable therapeutic entities which leverage the specificity of antibodies to selectively deliver cytotoxins to antigen-expressing targets such as cancer cells. However, current methods for their construction still suffer from a number of shortcomings. For instance, using a single modification technology to modulate the drug-to-antibody ratio (DAR) in integer increments while maintaining homogeneity and stability remains exceptionally challenging. Herein, we report a novel method for the generation of antibody conjugates with modular cargo loading from native antibodies. Our approach relies on a new class of disulfide rebridging linkers, which can react with eight cysteine residues, thereby effecting all-in-one bridging of all four interchain disulfides in an IgG1 antibody with a single linker molecule. Modification of the antibody with the linker in a 1 : 1 ratio enabled the modulation of cargo loading in a quick and selective manner through derivatization of the linker with varying numbers of payload attachment handles to allow for attachment of either 1, 2, 3 or 4 payloads (fluorescent dyes or cytotoxins). Assessment of the biological activity of these conjugates demonstrated their exceptional stability in human plasma and utility for cell-selective cytotoxin delivery or imaging/diagnostic applications.

The cylindrocyclophanes are a family of macrocyclic natural products reported to exhibit antibacterial activity. Little is known about the structural basis of this activity due to the challenges associated with their synthesis or isolation. We hypothesised that structural modification of the cylindrocyclophane scaffold could streamline their synthesis without significant loss of activity. Herein, we report a divergent synthesis of the cylindrocyclophane core enabling access to symmetrical macrocycles by means of a catalytic, domino cross-metathesis-ring-closing metathesis cascade, followed by late-stage diversification. Phenotypic screening identified several novel inhibitors of methicillin-resistant Staphylococcus aureus. The most potent inhibitor has a unique tetrabrominated [7,7]paracyclophane core with no known counterpart in nature. Together these illustrate the potential of divergent synthesis using catalysis and unbiased screening methods in modern antibacterial discovery.

All-syn fused cyclobutanes remain an elusive chemotype and thus present an interesting synthetic challenge. Herein, we report the successful application of Pd-catalysed C(sp3)-H arylation of cyclobutane compounds to generate all-syn heterobicyclic fragments using an innovative 'inside-out' approach. Through this strategy we generate a virtual collection of 90 fragments, which we demonstrate to have enhanced three-dimensionality and superior fragment-like properties compared to existing collections.

Site-selective modification of peptides and proteins has resulted in the development of a host of novel tools for the study of cellular systems or the synthesis of enhanced biotherapeutics. There is a need for useful methodologies that enable site-selective modification of native peptides or proteins, which is even more prevalent when modification of the biomolecule with multiple payloads is desired. Herein, we report the development of a novel dual functional divinylpyrimidine (dfDVP) platform that enables robust and modular modification of peptides, antibody fragments and antibodies. These biomacromolecules could be easily functionalised with a range of functional payloads (e.g. fluorescent dyes, cytotoxic warheads or cell-penetrating tags). Importantly, the dual functionalised peptides and antibodies demonstrated exquisite bioactivity in a range of in vitro cellular assays, showcasing the enhanced utility of these bioactive conjugates.

Inhibiting the aggregation of amyloid β (1-42) is a promising strategy for the development of disease-modifying Alzheimer’s disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of >200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic “nanoFLIM” assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid β self-assembly in vitro as well as in cellular and whole organism disease models.

N-Substituted dipropargylamines that are suitable as functionalized linkers for peptide stapling can be synthesized in one step under mild conditions from commercially available starting materials (41% to quantitative yield).

Bacterial infections caused by ‘superbugs’ are increasing globally, and conventional antibiotics are becoming less effective against these bacteria, such that we risk entering a post-antibiotic era. In recent years, antimicrobial peptides (AMPs) have gained significant attention for their clinical potential as a new class of antibiotics to combat antimicrobial resistance. In this review, we discuss several facets of AMPs including their diversity, physicochemical properties, mechanisms of action, and effects of environmental factors on these features. This review outlines various chemical synthetic strategies that have been applied to develop novel AMPs, including chemical modifications of existing peptides, semi-synthesis, and computer-aided design. We will also highlight novel AMP structures, including hybrids, antimicrobial dendrimers and polypeptides, peptidomimetics, and AMP–drug conjugates and consider recent developments in their chemical synthesis., This review discusses the diversity of structure and physicochemical properties of antimicrobial peptides and their derivatives, various chemical synthetic strategies that have been applied in their development, and how this links to their activity.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase’s biology, with wide-reaching implications for drug development.

A novel enzyme cleavable linker for antibody-drug conjugates is reported. The 3-O-sulfo-β-galactose linker is cleaved sequentially by two lysosomal enzymes - arylsulfatase A and β-galactosidase - to release the payload in targeted cells. An α-HER2 antibody-drug conjugate synthesised using this highly hydrophilic dual-cleavable linker exhibited excellent cytotoxicity and selectivity.

Peptides can offer the versatility needed for a successful oncology drug discovery approach. Peptide-drug conjugates (PDCs) are an emerging targeted therapeutic that present increased tumour penetration and selectivity. Despite these advantages, there are still limitations for the use of peptides as therapeutics exemplified through their slow progression to get into the clinic and limited oral bioavailability. New approaches to address these problems have been studied and successfully implemented to enhance the stability of peptides and their constructs. There is great promise for the future of PDCs with two molecules already on the market and many variations currently undergoing clinical trials, such as bicycle-toxin conjugates and peptide-dendrimer conjugates. This review summarises the entire process needed for the design and successful development of an oncology PDC including chemical and nanomaterial strategies to enhance peptide stability within circulation, the function of each component of a PDC construct, and current examples in clinical trials.

Dionysia tapetodes, a small cushion-forming mountainous evergreen in the Primulaceae, possesses a vast surface-covering of long silky fibres forming the characteristic “wooly” farina. This contrasts with some related Primula which instead possess a powdery farina. Using a combination of cell biology and analytical chemical techniques, we provide a detailed insight of wooly farina formation by glandular trichomes that produce a mixture of flavone and substituted flavone derivatives, including hydroxyflavones. Conversely, our analysis show that the powdery form consist almost entirely of flavone. The wooly farina in D. tapetodes is extruded through specific sites at the surface of the glandular head cell, characterised by a small complete gap in the plasma membrane, cell wall and cuticle. The data is consistent with formation and thread elongation occurring from within the cell. The putative mechanism of wool thread formation and its stability is discussed.