ACS Bio & Med Chem Au最新文献

Cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes constitute a large subclass of radical SAM (RS) enzymes that use Cbl to catalyze various types of reactions, the most common of which are methylations. Most Cbl-dependent RS enzymes contain an N-terminal Rossmann fold that aids Cbl binding. Recently, it has been demonstrated that the methanogenesis marker protein 10 (Mmp10) requires Cbl to methylate an arginine residue in the α-subunit of methyl coenzyme M reductase. However, Mmp10 contains a Cbl-binding domain in the C-terminal region of its primary structure that does not share significant sequence similarity with canonical RS Cbl-binding domains. Bioinformatic analysis of Mmp10 identified DUF512 (Domain of Unknown Function 512) as a potential Cbl-binding domain in RS enzymes. In this paper, four randomly selected DUF512-containing proteins from various organisms were overexpressed, purified, and shown to bind Cbl. X-ray crystal structures of DUF512-containing proteins from Clostridium sporogenes and Pyrococcus furiosus were determined, confirming their C-terminal Cbl-binding domains. The structure of the DUF512-containing protein from C. sporogenes is the first of an RS enzyme containing a PDZ domain. Its RS domain has an unprecedented β₃α₄ core, whereas most RS enzymes adopt a (βα)₆ core. The DUF512-containing protein from P. furiosus has no PDZ domain, but its RS domain also has an uncommon (βα)₅ core.

Mycophenolic acid (MPA), a natural compound, was modified to new MPA analogues via the classical method of silylation and esterification. Their cytotoxicity was evaluated in vitro on four osteosarcoma cancer cell lines (MNNG/HOS, U2OS, 143B, and SaOS-2) and human normal cells (hFOB 1.19). The most potent silicon-containing compound 2d (R¹ = TPS, R² = H) exhibited good cytotoxic activity against all osteosarcoma cancer cell lines with IC₅₀ values ranging from 0.64 to 2.27 μM and showing low cytotoxicity against normal cells. Further investigations revealed that compound 2d (R¹ = TPS, R² = H) displayed significant inhibition of IMPDH2 with K _{i app} 1.8 μM. Furthermore, molecular modeling studies were performed to investigate the binding affinity of 2d (R¹ = TPS, R² = H) which can effectively bind to critical amino acids of three proteins (vascular endothelial growth factor receptor 2; VEGFR-2, cyclin-dependent kinase 2; CDK2, inosine-5'-monophosphate dehydrogenase; IMPDH) involved in cancer therapy. This finding suggests that triphenylsilyl-MPA (TPS-MPA) analogue could serve as a promising starting point for developing new anticancer drugs for osteosarcoma.

Mycophenolic acid (MPA), a natural compound, was modified to new MPA analogues via the classical method of silylation and esterification. Their cytotoxicity was evaluated in vitro on four osteosarcoma cancer cell lines (MNNG/HOS, U2OS, 143B, and SaOS-2) and human normal cells (hFOB 1.19). The most potent silicon-containing compound 2d (R¹ = TPS, R² = H) exhibited good cytotoxic activity against all osteosarcoma cancer cell lines with IC₅₀ values ranging from 0.64 to 2.27 μM and showing low cytotoxicity against normal cells. Further investigations revealed that compound 2d (R¹ = TPS, R² = H) displayed significant inhibition of IMPDH2 with K_iapp 1.8 μM. Furthermore, molecular modeling studies were performed to investigate the binding affinity of 2d (R¹ = TPS, R² = H) which can effectively bind to critical amino acids of three proteins (vascular endothelial growth factor receptor 2; VEGFR-2, cyclin-dependent kinase 2; CDK2, inosine-5′-monophosphate dehydrogenase; IMPDH) involved in cancer therapy. This finding suggests that triphenylsilyl-MPA (TPS-MPA) analogue could serve as a promising starting point for developing new anticancer drugs for osteosarcoma.

Poly(ethylene glycol)-related immune responses have been a great concern regarding mRNA vaccination for the SARS-CoV2 virus, because PEG-lipids are an essential component for the lipid nanoparticles of mRNA vaccines. Meanwhile, no research has elucidated the mechanisms underlying hapten-like PEG-related immunogenicity. For the current study, we uncovered a process by which haptenic PEGs transition into immunogenic PEG-conjugates by means of ELISA and microfluidic diffusional sizing (MDS). We named the process “antigenicity extension.” Although PEGs exhibit specific interactions with anti-PEG antibodies, the specific interactions of PEGs with anti-PEG Abs are relatively weak. By contrast, we revealed that exposure of non-PEG moieties to the PEG-specific paratope greatly and directly contributes to PEG’s stable bindings through the specific interaction between PEG and anti-PEG antibodies by MDS measurements. This indicates that non-PEG moieties are directly involved in the molecular recognitions between PEG and the PEG-specific paratope to improve the affinity. Occurring antigenicity extension makes PEG-conjugates immunogenic by strengthening the affinity for PEG-specific paratopes. Thus, additional interactions at non-PEG moieties with the PEG-specific paratope are key to the transition of haptenic PEGs into immunogenic PEGs. To this extent, antigenicity extension is a commonly occurring phenomenon in the hapten-to-immunogen transitions occurring in both antigen–antibody interactions and ligand–receptor interactions.

Poly(ethylene glycol)-related immune responses have been a great concern regarding mRNA vaccination for the SARS-CoV2 virus, because PEG-lipids are an essential component for the lipid nanoparticles of mRNA vaccines. Meanwhile, no research has elucidated the mechanisms underlying hapten-like PEG-related immunogenicity. For the current study, we uncovered a process by which haptenic PEGs transition into immunogenic PEG-conjugates by means of ELISA and microfluidic diffusional sizing (MDS). We named the process "antigenicity extension." Although PEGs exhibit specific interactions with anti-PEG antibodies, the specific interactions of PEGs with anti-PEG Abs are relatively weak. By contrast, we revealed that exposure of non-PEG moieties to the PEG-specific paratope greatly and directly contributes to PEG's stable bindings through the specific interaction between PEG and anti-PEG antibodies by MDS measurements. This indicates that non-PEG moieties are directly involved in the molecular recognitions between PEG and the PEG-specific paratope to improve the affinity. Occurring antigenicity extension makes PEG-conjugates immunogenic by strengthening the affinity for PEG-specific paratopes. Thus, additional interactions at non-PEG moieties with the PEG-specific paratope are key to the transition of haptenic PEGs into immunogenic PEGs. To this extent, antigenicity extension is a commonly occurring phenomenon in the hapten-to-immunogen transitions occurring in both antigen-antibody interactions and ligand-receptor interactions.

The high throughput YESS 2.0 platform was used to screen a large library of SARS-CoV-2 M^pro variants in the presence of nirmatrelvir. Of the 100 individual most prevalent mutations identified in the screen and reported here, the most common were E166V, L27V, N142S, A173V, and Y154N, along with their various combinations. In vitro analysis revealed that resistance to nirmatrelvir for these individual mutations, as well as all of the combinations we analyzed, was accompanied by decreased catalytic activity with the native substrate. Importantly, the mutations we identified have not appeared as significantly enriched in SARS-CoV-2 M^pro sequences isolated from COVID-19 patients following the introduction of nirmatrelvir. We also analyzed three of the most common SARS-CoV-2 M^pro mutations that have been seen in patients recently, and only a measured increase in nirmatrelvir resistance was seen when the more recently appearing A285V is added to both P132H and K90R. Taken together, our results predict that resistance to nirmatrelvir will be slower to develop than expected based on experience with other viral protease inhibitors, perhaps due in part to the close structural correspondence between nirmatrelvir and SARS-CoV-2 M^pro's preferred substrates.

The high throughput YESS 2.0 platform was used to screen a large library of SARS-CoV-2 M^pro variants in the presence of nirmatrelvir. Of the 100 individual most prevalent mutations identified in the screen and reported here, the most common were E166V, L27V, N142S, A173V, and Y154N, along with their various combinations. In vitro analysis revealed that resistance to nirmatrelvir for these individual mutations, as well as all of the combinations we analyzed, was accompanied by decreased catalytic activity with the native substrate. Importantly, the mutations we identified have not appeared as significantly enriched in SARS-CoV-2 M^pro sequences isolated from COVID-19 patients following the introduction of nirmatrelvir. We also analyzed three of the most common SARS-CoV-2 M^pro mutations that have been seen in patients recently, and only a measured increase in nirmatrelvir resistance was seen when the more recently appearing A285V is added to both P132H and K90R. Taken together, our results predict that resistance to nirmatrelvir will be slower to develop than expected based on experience with other viral protease inhibitors, perhaps due in part to the close structural correspondence between nirmatrelvir and SARS-CoV-2 M^pro’s preferred substrates.