ABSTRACT
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.
ABSTRACT
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.
