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The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15–RBM39 interaction, we solved the DCAF15–DDB1–DDA1–indisulam–RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets. The crystal and cryo-electron microscopy structure analysis of the DCAF15–DDB1–DDA1–indisulam–RBM39 complex revealed the detailed mechanism of action of indisulam-induced RBM39 degradation and defined an α-helical degron motif in RBM39.

Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However, it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here, we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors, ATG8-like proteins, or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy, while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates. Using proximity-based screening, protein engineering, and structural analysis, this study describes the development of a p62-based biodegrader for the clearance of organelles and aggregated proteins by autophagy-targeted degradation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pulmonary arterial hypertension (PAH) has demonstrated multi-serotonin receptor dependent pathologies, characterized by increased tone (5-HT receptor) and complex lesions (SERT, 5-HT , 5-HT receptors) of the pulmonary vasculature together with right ventricular hypertrophy, ischemia and fibrosis (5-HT receptor). Selective inhibitors of individual signaling elements - SERT, 5-HT , 5HT , and combined 5-HT2 receptors, have all been tested clinically and failed. Thus, inhibition of tryptophan hydroxylase 1 (TPH1), the rate limiting step in 5-HT synthesis, has been suggested as a more broad, and thereby more effective, mode of 5-HT inhibition. However, selectivity over non-pathogenic enzyme family members, TPH2, phenylalanine hydroxylase, and tyrosine hydroxylase has hampered therapeutic development. Here we describe the site/sequence, biochemical, and biophysical characterization of a novel allosteric site on TPH1 through which selectivity over TPH2 and related aromatic amino acid hydroxylases is achieved. We demonstrate the mechanism of action by which novel compounds selectively inhibit TPH1 using surface plasma resonance and enzyme competition assays with both tryptophan ligand and BH4 co-factor. We demonstrate 15-fold greater potency within a human carcinoid cell line versus the most potent known TPH1/2 non-specific inhibitor. Lastly, we detail a novel canine system utilized to determine effective biologic inhibition of newly synthesized 5-HT. These findings are the first to demonstrate TPH1-selective inhibition and may pave the way to a truly effective means to reduce pathologic 5-HT and thereby treat complex remodeling diseases such as PAH.

The metabolic syndrome (MetSyn) is currently one of the biggest global health challenges because of its impact on public health. MetSyn includes the cluster of metabolic disorders including obesity, high blood pressure, hyperglycemia, high triglyceride levels, and hepatic steatosis. Together, these abnormalities increase the cardiovascular risk of individuals and pose a threat to healthcare systems worldwide. To better understand and address this complex issue, recent research has been increasingly focusing on unraveling the delicate interplay between metabolic disorders and the intestines and more specifically our gut microbiome. The gut microbiome entails all microorganisms inhabiting the gastrointestinal tract and plays a pivotal role in metabolic processes and overall health of its host. Emerging evidence proves an association between the gut microbiome composition and aspects of MetSyn, such as obesity. Understanding these relationships is crucial because they offer valuable insights into the mechanisms underlying development and progression of metabolic disorders and possible treatment options. Yet, how should we interpret this relationship? This review focuses on the interplay between the gut and MetSyn. In addition, we have reviewed the existing evidence of the gut microbiome and its association with and impact on metabolic disorders, in an attempt to understand the complex interactions and nature of this association. We also explored potential therapeutic options targeting the gut to modify metabolic disorders and obesity.

The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein. An inhibitor of the complement pathway of the innate immune system targets the human complement component 5 protein (C5) by binding to an interfacial pocket to prevent its proteolytic cleavage by the last enzyme of the complement pathway, C5 convertase.

Harnessing autophagy for targeted degradation is a promising extension to proteasome-based 18 targeted protein degradation because of the capacity and versatility of lysosomes to degrade large and 19 complex cargo, thus broadening the scope of therapeutic targets. While small-molecule degraders 20 recruiting the autophagy machinery to targets are starting to emerge, it remains unclear which component 21 of the autophagy lysosomal pathway is most efficacious to induce selective target degradation. Here, we 22 describe two orthogonal induced-proximity strategies to identify and prioritize autophagy effectors that are 23 sufficient to degrade organelles as well as soluble targets. We show that induced proximity of different 24 effectors such as autophagy cargo receptors, ATG8-like proteins or the kinases ULK1 and TBK1 are 25 sufficient to trigger mitophagy. In contrast, self-oligomerizing autophagy cargo receptors outperform ATG8-26 like effectors and autophagy-related kinases in clearing a soluble cytosolic protein. We further explore the 27 importance of avidity for targeted degradation via autophagy and reveal that the PB1 domain of p62 fused 28 to a LIR peptide is a minimal degron to induce the degradation of mitochondria as well as cytosolic proteins. 29 By developing a novel and highly selective intrabody against the autophagy cargo receptor p62 into a 30 heterobifunctional degrader, we demonstrate that recruitment of endogenous p62 is sufficient to clear 31 mitochondria. This biodegrader, however, is unable to induce degradation of soluble cytosolic proteins due 32 to its inhibitory effect on p62 self-oligomerization. Our study highlights the importance of avidity and 33 suggests that autophagy cargo receptors are attractive entry points for the development of 34 heterobifunctional degraders for complex targets such as organelles or protein aggregates.Competing Interest StatementZ.T, D.M., C.M., N.S., G.S., L.T., A.P., C.S.M., N.G., M.V., S.S.m A.L., J.-Y.C., L.F., P.H., C.B., F.V., S.G., M.M., F.F., G.M., and B.N. are employees of Novartis Pharma. B.G., L.E., and R.M. are former employees of Novartis.

The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Angstroms. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.

The environmental monitoring has been one of the priorities at the European and global scale due to the close relationship between the environmental pollution and the human health/socioeconomic development. In this field, the biosensors have been widely employed as cost-effective, fast, in situ, and real-time analytical techniques. The need of portable, rapid, and smart biosensing devices explains the recent development of biosensors with new transduction materials, obtained from nanotechnology, and for multiplexed pollutant detection, involving multidisciplinary experts. This review article provides an update on recent progress in biosensors for the monitoring of air, water, and soil pollutants in real conditions such as pesticides, potentially toxic elements, and small organic molecules including toxins and endocrine disrupting chemicals.

A bstract Four independent dimension-eight operators give rise to anomalous neutral triple gauge boson interactions, one CP-even and three CP-odd. Only the CP-even operator interferes with the Standard Model for the production of a pair of on-shell neutral bosons. However, the effects are found to be tiny due mainly to the mismatch of the Z boson polarization between the productions from the SM and the new operator.