Correction: Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis.

Correction for 'Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis' by Maurício Morais et al., Org. Biomol. Chem., 2017, 15, 2947-2952, DOI: .

Controlled coupling of an ultrapotent auristatin warhead to cetuximab yields a next-generation antibody-drug conjugate for EGFR-targeted therapy of KRAS mutant pancreatic cancer.

BACKGROUND: Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC. METHODS: In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens. RESULTS: Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells. CONCLUSIONS: Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

A Plug-and-Play Approach for the De Novo Generation of Dually Functionalized Bispecifics.

Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics on the market interact with only one target, thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics has emerged: bispecific antibodies. Bispecific formation using chemical methods is rare and low-yielding and/or requires a large excess of one of the two proteins to avoid homodimerization and heterogeneity. In order for chemically prepared bispecifics to deliver their full potential, high-yielding, modular, and reliable cross-linking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chemical generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalization of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalized bispecifics with controlled loading in a modular and convergent manner.

Post-translational site-selective protein backbone α-deuteration.

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-1H → C-2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a 'carba-Swern' mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C-D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine.

Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones.

Due to their exquisite cysteine-selectivity, excellent stability, and ability to functionally rebridge disulfide bonds, dibromopyridazinediones are emerging as an exciting new class of bioconjugation reagents, particularly in the field of antibody conjugation. Despite this, relatively little work has been performed on the optimisation of their synthesis and subsequent reaction with immunoglobulins. Herein we present a novel synthetic route towards functionalised dibromopyridazinediones, proceeding via an isolatable dibromopyridazinedione-NHS ester. Reaction of this activated intermediate with a variety of amines produces functional dibromopyridazinediones in good to excellent yields. The disulfide rebridging capacity of these reagents was optimised on the clinically relevant IgG1 trastuzumab, resulting in a general method which allows for the generation of site-selectively modified native trastuzumab with over 90% homogeneity (no disulfide scrambling) without the need for protein engineering or enzymatic conjugation.

Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis.

Disulfide bridging offers a convenient approach to generate site-selective antibody conjugates from native antibodies. To optimise the reagents available to achieve this strategy, we describe here the use of dibromomaleimides designed to undergo accelerated post-conjugation hydrolysis. Conjugation and hydrolysis, which serve to 'lock' the conjugates as robustly stable maleamic acids, is achieved in just over 1 h. This dramatic acceleration is also shown to infer significant improvements in homogeneity, as demonstrated by mass spectrometry analysis.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Identification of an active metabolite of PAR-1 antagonist RWJ-58259 and synthesis of analogues to enhance its metabolic stability.

The discontinuation of PAR-1 antagonist RWJ-58259 beyond use as a biological probe is most likely due to it's short half-life in vivo. However, retention of significant in vivo activity beyond the point where most of the RWJ-58259 had been consumed implies the generation of an active metabolite. Herein we describe the biological activity of a predicted metabolite of RWJ-58259 and the synthesis of analogues designed to enhance the metabolic stability of RWJ-58259.

Synthesis of novel and potent vorapaxar analogues.

Vorapaxar is a first-in-class PAR-1 antagonistic drug based on the ent-himbacine scaffold. Detailed in this article are enantioselective and racemic routes to various novel vorapaxar analogues. Biological testing revealed these compounds to have moderate to excellent potencies against PAR-1 with the most potent analogue demonstrating an IC50 of 27 nM.

A novel synthetic chemistry approach to linkage-specific ubiquitin conjugation.

Ubiquitination is of great importance as the post-translational modification of proteins with ubiquitin, or ubiquitin chains, facilitates a number of vital cellular processes. Herein we present a facile method of preparing various ubiquitin conjugates under mild conditions using michael acceptors based on dibromo-maleimides and dibromo-pyridazinediones.

A platform for efficient, thiol-stable conjugation to albumin's native single accessible cysteine.

Herein we report the use of bromomaleimides for the construction of stable albumin conjugates via conjugation to its native, single accessible, cysteine followed by hydrolysis. Advantages over the classical maleimide approach are highlighted in terms of quantitative hydrolysis and absence of undesirable retro-Michael deconjugation.

Pharmacological inhibition of DDAH1 improves survival, haemodynamics and organ function in experimental septic shock.

The aim of the present study was to investigate the therapeutic effects of pharmacological inhibition of DDAH1 (dimethylarginine dimethylaminohydrolase 1), an enzyme that metabolizes endogenously produced nitric oxide synthase inhibitors, principally ADMA (asymmetric dimethylarginine). The present study employs a series of rodent models to evaluate the effectiveness a DDAH1-selective inhibitor (L-257). Short-term models involved the development of endotoxaemia using lipopolysaccharide and long-term models involved the intraperitoneal administration of faecal slurry. In order to generate the most relevant model possible, following induction of severe sepsis, animals received appropriate fluid resuscitation and in some models vasopressor therapy. The effects of L-257 on survival, haemodynamics and organ function were subsequently assessed. Survival was significantly longer in all L-257 treatment groups (P<0.01) and no adverse effects on haemodynamics and organ function were observed following L-257 administration to either animals with sepsis or naïve animals. Haemodynamic performance was preserved and the noradrenaline dose required to maintain target blood pressure was reduced in the treated animals (P<0.01). Animals receiving L-257 had significantly increased plasma ADMA concentrations. Plasma nitrite/nitrate was reduced as was severity of sepsis-associated renal dysfunction. The degree of tachycardia was improved as were indices of tissue and microvascular perfusion. The results of the present study show that the selective DDAH-1 inhibitor L-257 improved haemodynamics, provided catecholamine sparing and prolonged survival in experimental sepsis. Further studies will determine its potential utility in human septic shock.

Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment.

The rapidly increasing interest in the synthesis of antibody-drug conjugates as powerful targeted anticancer agents demonstrates the growing appreciation of the power of antibodies and antibody fragments as highly selective targeting moieties. This targeting ability is of particular interest in the area of photodynamic therapy, as the applicability of current clinical photosensitizers is limited by their relatively poor accumulation in target tissue in comparison to healthy tissue. Although synthesis of porphyrin-antibody conjugates has been previously demonstrated, existing work in this area has been hindered by the limitations of conventional antibody conjugation methods. This work describes the attachment of azide-functionalized, water-soluble porphyrins to a tratuzumab Fab fragment via a novel conjugation methodology. This method allows for the synthesis of a homogeneous product without the loss of structural stability associated with conventional methods of disulfide modification. Biological evaluation of the synthesized conjugates demonstrates excellent selectivity for a HER2 positive cell line over the control, with no dark toxicity observed in either case.

Cyclisation reactions of N-cinnamoyl-9-aminoanthracenes.

N-Cinnamoyl-9-aminoanthracenes cyclise with PPA or triflic acid to form novel 2-azahexacyclo[10.6.6.0(1,5).0(6,11).0(13,18).0(19,24)]tetracosa-6(11),7,9,13,15,17,19(24),20,22-nonaen-3-ones. In contrast, both N-cinnamoyl-N-methyl-9-(2-aminomethyl)anthracene and N-cinnamoyl-9-(2-aminoethyl)anthracene undergo an intramolecular Diels-Alder cycloaddition.

Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging.

The advent of Adcetris™ and Kadcyla™, two recently FDA-approved antibody-drug conjugates (ADCs), in the clinic has had a major impact on the treatment of lymphoma and breast cancer patients, respectively, worldwide. Despite these successes many new ADCs fail at various stages of development, often due to shortcomings in the methods used for their assembly. To address this problem we have developed next generation maleimides (NGMs), which specifically re-bridge reduced interchain disulfide bonds and allow the efficient conjugation of small molecules to antibodies, without the need for engineering of the target antibody. The method is site-specific and generates near homogeneous products in good yields. Moreover, adjustment of the reaction conditions allows control of the conjugation in terms of stoichiometry (drug-loading) and site selectivity. Using this method we prepared a series of ADCs from trastuzumab and doxorubicin (DOX) with a controlled drug-to-antibody ratio (DAR) of 1, 2, 3 and 4. All of these constructs were fully active by ELISA and had more than 90% of re-bridged disulfide bonds by CE-SDS when compared to clinical grade antibody. Furthermore, digest experiments of the DAR 2 material revealed that almost all of the drug had been targeted to the Fab arms of the antibody. Thus, NGMs offer a flexible and simple platform for the controlled assembly of ADCs from an antibody.

Bromo- and thiomaleimides as a new class of thiol-mediated fluorescence 'turn-on' reagents.

Bromo- and thiomaleimides are shown to serve as highly effective quenchers of a covalently attached fluorophore. Reactions with thiols that lead to removal of the maleimide conjugation, or detachment of the fluorophore from the maleimide, result in 'turn-on' of the fluorescence. These reagents thus offer opportunities in thiol sensing and intracellular reporting.

The triflic acid-mediated cyclization reactions of N-cinnamoyl-1-naphthylamines.

N-cinnamoyl-1-naphthylamines undergo a cyclization reaction with triflic acid to form 4-phenyl-3,4-dihydro-1H-naphth[1,8-bc]azepin-2-ones and 4-phenyl-3,4-dihydro-1H-benzo[h]quinolin-2-ones. However, the N-benzyl analogues also undergo a unique cascade reaction to form novel heptacyclic structures via a 1,2-addition followed by a 4-addition to the naphthalene. With an electron-rich N-benzyl substituent, the heptacycle is the sole product.

Metal-free, hydroacylation of C=C and N=N bonds via aerobic C-H activation of aldehydes, and reaction of the products thereof.

In this report, a thorough evaluation of the use of aerobically initiated, metal-free hydroacylation of various C=C and N=N acceptor molecules with a wide range of aldehydes is presented. The aerobic-activation conditions that have been developed are in sharp contrast to previous conditions for hydroacylation, which tend to use transition metals, peroxides that require thermal or photochemical degradation, or N-heterocyclic carbenes. The mildness of the conditions enables a number of reactions involving sensitive reaction partners and, perhaps most significantly, allows for α-functionalised chiral aldehydes to undergo radical-based hydroacylation with complete retention of optical purity. We also demonstrate how the resulting hydroacylation products can be transformed into other useful intermediates, such as γ-keto-sulfonamides, sultams, sultones, cyclic N-sulfonyl imines and amides.

Reversible protein affinity-labelling using bromomaleimide-based reagents.

Reversible protein biotinylation is readily affected via conjugation with a bromomaleimide-based reagent followed by reductive cleavage. The intermediate biotinylated protein constructs are stable at physiological temperature and pH 8.0. Quantitative reversibility is elegantly delivered under mild conditions of using a stoichiometric amount of a bis-thiol, thus providing an approach that will be of general interest in chemical biology and proteomics.

A mild synthesis of N-functionalised bromomaleimides, thiomaleimides and bromopyridazinediones.

Bromomaleimides are useful building blocks in synthesis and powerful reagents for the selective chemical modification of proteins. A mild new synthesis of these reagents is described, along with the convenient transferability of the approach to dithiomaleimides and bromopyridazinediones.