A RIPK1-specific PROTAC degrader achieves potent antitumor activity by enhancing immunogenic cell death.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a critical stress sentinel that coordinates cell survival, inflammation, and immunogenic cell death (ICD). Although the catalytic function of RIPK1 is required to trigger cell death, its non-catalytic scaffold function mediates strong pro-survival signaling. Accordingly, cancer cells can hijack RIPK1 to block necroptosis and evade immune detection. We generated a small-molecule proteolysis-targeting chimera (PROTAC) that selectively degraded human and murine RIPK1. PROTAC-mediated depletion of RIPK1 deregulated TNFR1 and TLR3/4 signaling hubs, accentuating the output of NF-κB, MAPK, and IFN signaling. Additionally, RIPK1 degradation simultaneously promoted RIPK3 activation and necroptosis induction. We further demonstrated that RIPK1 degradation enhanced the immunostimulatory effects of radio- and immunotherapy by sensitizing cancer cells to treatment-induced TNF and interferons. This promoted ICD, antitumor immunity, and durable treatment responses. Consequently, targeting RIPK1 by PROTACs emerges as a promising approach to overcome radio- or immunotherapy resistance and enhance anticancer therapies.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype.

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an Nε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

Novel irreversible peptidic inhibitors of transglutaminase 2.

Transglutaminase 2 (TG2), also referred to as tissue transglutaminase, plays crucial roles in both protein crosslinking and cell signalling. It is capable of both catalysing transamidation and acting as a G-protein, these activities being conformation-dependent, mutually exclusive, and tightly regulated. The dysregulation of both activities has been implicated in numerous pathologies. TG2 is expressed ubiquitously in humans and is localized both intracellularly and extracellularly. Targeted TG2 therapies have been developed but have faced numerous hurdles including decreased efficacy in vivo. Our latest efforts in inhibitor optimization involve the modification of a previous lead compound's scaffold by insertion of various amino acid residues into the peptidomimetic backbone, and derivatization of the N-terminus with substituted phenylacetic acids, resulting in 28 novel irreversible inhibitors. These inhibitors were evaluated for their ability to inhibit TG2 in vitro and their pharmacokinetic properties, and the most promising candidate 35 (k inact/K I = 760 × 103 M-1 min-1) was tested in a cancer stem cell model. Although these inhibitors display exceptional potency versus TG2, with k inact/K I ratios nearly ten-fold higher than their parent compound, their pharmacokinetic properties and cellular activity limit their therapeutic potential. However, they do serve as a scaffold for the development of potent research tools.

Structure-activity relationships of N-terminal variants of peptidomimetic tissue transglutaminase inhibitors.

Tissue transglutaminase (TG2) is a multifunctional protein that catalyses protein crosslinking in the extracellular matrix, and functions as an intracellular G-protein. While both activities have been associated with human diseases, its role as a G-protein has been linked to cancer stem cell survival and maintenance of a metastatic phenotype. Recently we have shown that targeted covalent inhibitors (TCIs) can react selectively with the enzyme active site of TG2, to allosterically abolish its ability to bind GTP. In the present work, we focused on the variation of the N-terminal group of these peptidomimetic inhibitors, in order to enhance efficiency, while reducing log P and the number of rotatable bonds. This approach led to the synthesis and evaluation of 41 novel inhibitors, some of which had greatly improved efficiency and affinity for TG2 (e.g. TCI 72: KI = 1.0 µM, kinact/KI = 4.4 × 105 M-1 min-1). Molecular modelling provided a hypothetical binding mode for these TCIs. The most efficient inhibitors were evaluated further and shown to have excellent isozyme selectivity, to block GTP binding, and to have improved pharmacokinetic properties, as expected. Their biological activity was also confirmed, in a cellular invasion assay, although with less potency than expected.

Design, synthesis and evaluation of tryptophan analogues as tool compounds to study IDO1 activity.

The metabolism of l-tryptophan to N-formyl-l-kynurenine by indoleamine-2,3-dioxygenase 1 (IDO1) is thought to play a critical role in tumour-mediated immune suppression. Whilst there has been significant progress in elucidating the overall enzymatic mechanism of IDO1 and related enzymes, key aspects of the catalytic cycle remain poorly understood. Here we report the design, synthesis and biological evaluation of a series of tryptophan analogues which have the potential to intercept putative intermediates in the metabolism of 1 by IDO1. Functionally-relevant binding to IDO1 was demonstrated through enzymatic inhibition, however no IDO1-mediated metabolism of these compounds was observed. Subsequent T m-shift analysis shows the most active compound, 17, exhibits a distinct profile from known competitive IDO1 inhibitors, with docking studies supporting the hypothesis that 17 may bind at the recently-discovered Si site. These findings provide a start-point for development of further mechanistic probes and more potent tryptophan-based IDO1 inhibitors.