Wiita, E. | Search bioRxiv

bioRxiv2023

Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1 via a comprehensive screening funnel

Sterile alpha motif and histidine-aspartic acid domain containing protein-1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase central to cellular nucleotide pool homeostasis. Recent literature has also demonstrated how SAMHD1 can detoxify chemotherapy metabolites thereby controlling their clinical responses. To further understand SAMHD1 biology and to investigate the potential of targeting this enzyme as a neoadjuvant to existing chemotherapies we set out to discover selective small molecule-based inhibitors of SAMHD1. Here we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for further characterisation of the screen output. The identified hit compound TH6342 and its analogues, accompanied by their inactive negative control analogue TH7126, demonstrated specific, low M potency in inhibiting the hydrolysis of both natural substrates and nucleotide analogue therapeutics, shown using complementary enzyme-coupled and direct enzymatic activity assays. Their mode of inhibition was subsequently detailed by coupling kinetic studies with thermal shift assays, where TH6342 and analogues were shown to engage with pre-tetrameric SAMHD1 and deter the oligomerisation and allosteric activation of SAMHD1 without occupying nucleotide binding pockets. We further outline the development and application of multiple cellular assays for assessing cellular target engagement and associated functional effects, including CETSA and an in-cell dNTP hydrolase activity assay, which highlighted future optimisation strategies of this chemotype. In summary, with a novel mode of inhibition, TH6342 and analogues broaden the set of tool compounds available in deciphering SAMHD1 enzymology and functions, and furthermore, the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/524275v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dc706forg.highwire.dtl.DTLVardef@59a9b4org.highwire.dtl.DTLVardef@9473e2org.highwire.dtl.DTLVardef@4427dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Selenium Silk Nanostructured Films for Antifungal and Antibacterial Treatments

The rapid emergence of drug-resistant bacteria and fungi poses a threat for healthcare worldwide. The development of novel effective small molecule therapeutic strategies in this space has remained challenging. An orthogonal approach, therefore, is to explore biomaterials with physical modes of action which have the potential to generate antimicrobial activity and in some cases even prevent antimicrobial resistance. Here, to this effect, we describe an approach for forming silk-based films that contain embedded selenium nanoparticles. We show that these materials exhibit both antibacterial and antifungal properties while crucially also remaining highly biocompatible and non-cytotoxic towards mammalian cells. By incorporating the nanoparticles into silk films, the protein scaffold acts in a two-fold manner; it protects the mammalian cells from the cytotoxic effects of the bare nanoparticles, while also providing a template for bacterial and fungal eradication. A range of hybrid inorganic/organic films were produced and an optimum concentration was found, which allowed for both high bacterial and fungal death while also exhibiting low mammalian cell cytotoxicity. Such films can thus pave the way for next generation antimicrobial materials for applications such as wound healing and as agents against topical infections, with the added benefit that bacteria and fungi are unlikely to develop antimicrobial resistance to these hybrid materials.

microbiology↗