Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle.

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a KD of 14 µM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells.

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a k inact /K I of 1.0 x 10 6 M -1 s -1 and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression.

Characterization of Daboia russelii and Naja naja venom neutralizing ability of an undocumented indigenous medication in Sri Lanka.

BACKGROUND: Indigenous medicinal practice in Sri Lanka talks about powerful compounds extracted from native plants for treating venomous snake bites which are hardly documented in literature but are used by the indigenous doctors for thousand years. OBJECTIVE: We screened the neutralizing ability of a herbal preparation practiced in indigenous medicine of Sri Lanka, consisting of Sansevieria cylindrica, Jatropha podagrica and Citrus aurantiifolia, for its ability to neutralize venom toxins of Naja naja (Common Cobra) and Daboia russelii (Russell's viper). MATERIALS AND METHODS: The venom toxicity was evaluated using a 5-day old chicken embryo model observing the pathophysiology and the mortality for six hours, in the presence or absence of the herbal preparation. The known toxin families to exist in snake venom, such as Phospholipase A2, Snake venom Metalloprotease, were evaluated to understand the mechanism of venom neutralizing ability of the herbal preparation. RESULTS: The LD50 of D. russelii venom, as measured using the 5-day old chicken embryo model, was 4.8 ± 0.865 ug (R2 = 84.8%, P = 0.079). The pre-incubation of venom with the herbal preparation increased the LD50 of D. russelii venom to 17.64 ± 1.35 µg (R2 = 81.0%, P = 0.100), showing a clear neutralizing action of D. russelii venom toxicity by the herbal medicine. Whereas the pre-incubation of venom with the 1× venom neutralizing dose of commercially available polyvalent anti-venom serum shifted the LD50 venom only up to 5.5 ± 1.35 µg (R2 = 98.8%, P = 0.069). In the presence of the herbal preparation, Phospholipase A2 activity of D. russelii venom was significantly reduced from 9.2 × 10-3 mM min-1 to 8.0 × 10-3 mM min-1 and that of N. naja from 2.92 × 10-2 mM min-1 to 0.188 × 10-2 mM min-1. Further, the pre-incubation of N. naja venom with the herbal preparation significantly reduced its Metalloprotease activity from 0.069 units min-1 to 0.019 units min-1. CONCLUSION: The herbal preparation shows a clear neutralizing action against the toxicities of D. russelii and N. naja venoms demonstrating the potential to be used as a plant based antidote for snake envenomation.