Design and Evaluation of PROTACs Targeting Acyl Protein Thioesterase 1

PROTAC linker design remains mostly an empirical task. We employed the PRosettaC computational software in the design of sulfonyl-fluoride-based PROTACs targeting acyl protein thioesterase 1 (APT1). The software efficiently generated ternary complex models from empirically-designed PROTACs and suggested alkyl linkers to be the preferred type of linker to target APT1. Western blotting analysis revealed efficient degradation of APT1 and activity-based protein profiling showed remarkable selectivity of an alkyl linker-based PROTAC amongst serine hydrolases. Collectively, our data suggests that combining PRosettaC and chemoproteomics can effectively assist in triaging PROTACs for synthesis and providing early data on their potency and selectivity.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 101022421 (L.A.R.C.). D.Z. was supported by the Blavatnik Cambridge Fellowship, funded by the Blavatnik Family Foundation. This study was supported by Fundação para a Ciência e a Tecnologia (FCT, Portugal) through project SFRH/BD/143583/2019 (B.B.S)

Cathepsin B Processing is Required for the in vivo Efficacy of Albumin-Drug Conjugates

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigate whether cathepsin B cleavage of a valine-citrulline [VC(S)] containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we use an engineered version of human serum albumin, Veltis® High Binder II (HBII), which has enhanced binding to the neonatal Fc receptor (FcRn) to improve drug-release upon binding and FcRn-mediated recycling. The linker–payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogenous and plasma stable conjugates that retained FcRn binding. Two caa–linker–MMAE reagents were synthesised — one with a cleavable [VC(S)] linker and one with a non-cleavable [VC(R)] linker — to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective anti-tumour activity. The conjugates equipped with the cleavable [VC(S)] linker had potent anti-tumour activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalisation. In addition to the pronounced anti-tumour activity of the albumin conjugates in vivo, we also demonstrated their preferable tumour biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumours

Adaptive Optimization of Chemical Reactions with Minimal Experimental Information

Optimizing reaction conditions is cornerstone in synthetic chemistry and connected to expert chemistry knowledge, as well as laborious exploration of reaction parameters. To automate reaction condition optimizations and augment chemical intuition, we developed adaptive machine intelligence to navigate search spaces. Our approach (LabMate.AI) employs an interpretable algorithm and requires only 0.03–0.04% of all search space as input data. LabMate.AI optimizes many reaction parameters simultaneously, with minimal computational resources and time. In proof-of-concept studies with distinct goals, we demonstrate how LabMate.AI can identify optimal conditions for an Ugi and a C–N cross-coupling reaction in a competitive manner relative to human experts without requiring specialized chemistry equipment and using orders of magnitude less data than related approaches. LabMate.AI affords quantitative and interpretable reactivity insights, is rapidly deployable and autonomously formalizes chemical intuition, thereby providing an innovative framework towards informed and automated experiment optimization and democratization of synthetic chemistry

The Impact of Activity-based Protein Profiling in Malaria Drug Discovery.

Activity-based protein profiling (ABPP) is an approach used at the interface of chemical biology and proteomics that uses small molecular probes to provide dynamic fingerprints of enzymatic activity in complex proteomes. Malaria is a disease caused by Plasmodium parasites with a significant death burden and for which new therapies are actively being sought. Here, we compile the main achievements from ABPP studies in malaria and highlight the probes used and the different downstream platforms for data analysis. ABPP has excelled at studying Plasmodium cysteine proteases and serine hydrolase families, the targeting of the proteasome and metabolic pathways, and in the deconvolution of targets and mechanisms of known antimalarials. Despite the major impact in the field, many antimalarials and enzymatic families in Plasmodium remain to be studied, which suggests ABPP will be an evergreen technique in the field

Structural insights into TRPV2 activation by small molecules

Transient receptor potential vanilloid 2 (TRPV2) is involved in many critical physiological and pathophysiological processes, making it a promising drug target. Here we present cryo-electron microscopy (cryo-EM) structures of rat TRPV2 in lipid nanodiscs activated by 2-aminoethoxydiphenyl borate (2-APB) and propose a TRPV2-specific 2-ABP binding site at the interface of S5 of one monomer and the S4-S5 linker of the adjacent monomer. In silico docking and electrophysiological studies confirm the key role of His521 and Arg539 in 2-APB activation of TRPV2. Additionally, electrophysiological experiments show that the combination of 2-APB and cannabidiol has a synergetic effect on TRPV2 activation, and cryo-EM structures demonstrate that both drugs were able to bind simultaneously. Together, our cryo-EM structures represent multiple functional states of the channel, providing a native picture of TRPV2 activation by small molecules and a structural framework for the development of TRPV2-specific activators

Azabicyclic vinyl sulfones for residue-specific dual protein labelling.

We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are moderately resistant to retro Diels-Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1-5 equiv. of reagent in NaPi pH 7.0, room temperature, 30 min). The resulting thioether-linked conjugates were stable and retained the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein could be fluorescently labelled through an inverse electron demand Diels-Alder reaction in cells to allow selective apoptosis imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine

Stable Pyrrole-Linked Bioconjugates through Tetrazine-Triggered Azanorbornadiene Fragmentation.

We have developed an azanorbornadiene bromovinyl sulfone reagent that allows cysteine-selective bioconjugation. Subsequent reaction with dipyridyl tetrazine led to bond-cleavage and formation of a pyrrole-linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene-tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro-Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site-selective bioconjugation followed by bioorthogonal bond-cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which lends it to the routine preparation of protein conjugates for chemical biology studies

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors