Biology subjects

Snee, M.

Publications and source records attributed to Snee, M..

3 recordsLinked to original sources

bioRxiv2024

Fragment-based development of small molecule inhibitors targeting Mycobacterium tuberculosis cholesterol metabolism

Mycobacterium tuberculosis (Mtb) is the worlds most deadly infectious pathogen and new drugs are urgently required to combat the emergence of multi-(MDR) and extensively-(XDR) drug resistant strains. The bacterium specifically upregulates sterol uptake pathways in infected macrophages and the metabolism of host-derived cholesterol is essential for Mtbs long-term survival in vivo. Here, we report the development of antitubercular small molecules that inhibit the Mtb cholesterol oxidases CYP125 and CYP142, which catalyze the initial step of cholesterol metabolism. An efficient biophysical fragment screen was used to characterize the structure-activity relationships of CYP125 and CYP142, and identify a non-azole small molecule 1a that can bind to the heme cofactor of both enzymes. A structure-guided fragment-linking strategy was used to optimize the binding affinity of 1a, yielding a potent dual CYP125/142 inhibitor 5m (KD CYP125/CYP142 = 0.04/0.16 {micro}M). Compound 5m potently inhibits the catalytic activity of CYP125 and CYP142 in vitro (KI values < 0.1 {micro}M), and rapidly depletes Mtb intracellular ATP (IC50 = 0.15 {micro}M). The compound has antimicrobial activity against both drug susceptible and MDR Mtb (MIC99 values 0.4 - 1.5 {micro}M) in extracellular assays, and inhibits the growth of Mtb in human macrophages (MIC = 1.7 {micro}M) with good selectivity over mammalian cytotoxicity (LD50 [≥] 50 {micro}M). The combination of small molecule inhibitors and structural data reported here provide useful tools to study the role of cholesterol metabolism in Mtb and are a promising step towards novel antibiotics targeting bioenergetic pathways, which could be used to help combat MDR-TB.

pharmacology and toxicology↗

bioRxiv2024

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-alpha-inhibitor regulate crosslinking of the hyaluronan matrix

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter--inhibitor (II) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC*HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric Half-PTX3 and a construct missing the 24 N-terminal residues ({Delta}1-24-PTX3). SAXS determined a length of [~]520 [A] for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC*HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC*HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC*HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

biochemistry↗

bioRxiv2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

biophysics↗