Efficient and selective antibody modification with functionalised divinyltriazines.

A highly efficient disulfide rebridging strategy for the modification of monoclonal antibodies with substituted divinyltriazine linkers is reported. The reaction proceeds efficiently under mild conditions with near stoichiometric quantities of linker. This method of conjugation yields serum stable antibody conjugates with a controlled payload loading of 4.

Using Peptidomimetics and Constrained Peptides as Valuable Tools for Inhibiting Protein⁻Protein Interactions.

Protein⁻protein interactions (PPIs) are tremendously important for the function of many biological processes. However, because of the structure of many protein⁻protein interfaces (flat, featureless and relatively large), they have largely been overlooked as potential drug targets. In this review, we highlight the current tools used to study the molecular recognition of PPIs through the use of different peptidomimetics, from small molecules and scaffolds to peptides. Then, we focus on constrained peptides, and in particular, ways to constrain α-helices through stapling using both one- and two-component techniques.

Robust asymmetric synthesis of unnatural alkenyl amino acids for conformationally constrained α-helix peptides.

The efficient asymmetric synthesis of unnatural alkenyl amino acids required for peptide 'stapling' has been achieved using alkylation of a fluorine-modified Ni(II) Schiff base complex as the key step.

A recombinant approach for stapled peptide discovery yields inhibitors of the RAD51 recombinase.

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.

Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes.

Targeting the lysine deacetylase activity of class I histone deacetylases (HDACs) is potentially beneficial for the treatment of several diseases including human immunodeficiency virus (HIV) infection, Alzheimer's disease, and various cancers. It is therefore important to understand the function and mechanism of action of these enzymes. Class I HDACs act as catalytic components of seven large, multiprotein corepressor complexes. Different HDAC corepressor complexes have specific, nonredundant roles in the cell. It is likely that their specific functions are at least partly influenced by the substrate specificity of the complexes. To address this, we developed chemical tools to probe the specificity of HDAC complexes. We assessed a library of acetyl-lysine-containing substrate peptides and hydroxamic acid-containing inhibitor peptides against the full range of class I HDAC corepressor complexes. The results suggest that site-specific HDAC corepressor complex activity is driven in part by the recognition of the primary amino acid sequence surrounding a particular lysine position in the histone tail.

Divergent Synthesis of Novel Cylindrocyclophanes that Inhibit Methicillin-Resistant Staphylococcus aureus (MRSA).

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.

Synthesis and Reactivity of a Bis-Strained Alkyne Derived from 1,1'-Biphenyl-2,2',6,6'-tetrol.

The novel "double strained alkyne" 3 has been prepared and evaluated in strain-promoted azide-alkyne cycloaddition reactions with azides. The X-ray crystallographic structure of 3, which was prepared in one step from 1,1'-biphenyl-2,2',6,6'-tetrol 4, reveals the strained nature of the alkynes. Dialkyne 3 undergoes cycloaddition reactions with a number of azides, giving mixtures of regiosiomeric products in excellent yields. The monoaddition products were not observed or isolated from the reactions, suggesting that the second cycloaddition proceeds at a faster rate than the first, and this is supported by molecular modeling studies. Dialkyne 3 was successfully employed for "peptide stapling" of a p53-based diazido peptide, whereby two azides are bridged to give a product with a stabilized conformation.

Macrocyclisation and functionalisation of unprotected peptides via divinyltriazine cysteine stapling.

We report a novel divinyltriazine linker for the stapling of two cysteine residues to form macrocyclic peptides from their unprotected linear counterparts. The stapling reaction occurred rapidly under mild conditions on a range of unprotected peptide sequences. The resulting constrained peptides displayed greater stability in a serum stability assay when compared to their linear counterparts.

Insights into the activation mechanism of class I HDAC complexes by inositol phosphates.

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.