Systemic whole transcriptome analysis identified underlying molecular characteristics and regulatory networks implicated in the retina following optic nerve injury.

Optic nerve injuries are severely disrupt the structural and functional integrity of the retina, often leading to visual impairment or blindness. Despite the profound impact of these injuries, the molecular mechanisms involved remain poorly understood. In this study, we performed a comprehensive whole-transcriptome analysis of mouse retina samples after optic nerve crush (ONC) to elucidate changes in gene expression and regulatory networks. Transcriptome analysis revealed a variety of molecular alterations, including 256 mRNAs, 530 lncRNAs, and 37 miRNAs, associated with metabolic, inflammatory, signaling, and biosynthetic pathways in the injured retina. The integrated analysis of co-expression and protein-protein interactions identified an active interconnected module comprising 5 co-expressed proteins (Fga, Serpina1a, Hpd, Slc38a4, and Ahsg) associated with the complement and coagulation cascades. Finally, 5 mRNAs (Fga, Serpinala, Hpd, Slc38a4, and Ahsg), 2 miRNAs (miR-671-5p and miR-3057-5p), and 6 lncRNAs (MSTRG. 1830.1, Gm10814, A530013C23Rik, Gm40634, MSTRG.9514.1, A330023F24Rik) were identified by qPCR in the injured retina, and some of them were validated as critical components of a ceRNA network active in 661W and HEK293T cells through dual-luciferase reporter assays. In conclusion, our study provides comprehensive insight into the complex and dynamic biological mechanisms involved in retinal injury responses and highlights promising potential targets to enhance neuroprotection and restore vision.

Discovery of natural catechol derivatives as covalent SARS-CoV-2 3CLpro inhibitors.

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a threat to public health, and extensive research by scientists worldwide has also prompted the development of antiviral therapies. The 3C-like protease (3CLpro) is critical for SARS-CoV-2 replication and acts as an effective target for drug development. To date, numerous of natural products have been reported to exhibit inhibitory effects on 3CLpro, which encourages us to identify other novel inhibitors and elucidate their mechanism of action. In this study, we first screened an in-house compound library of 101 natural products using FRET assay, and found that oleuropein showed good inhibitory activity against SARS CoV-2 3CLpro with an IC50 value of 4.18 µM. Further studies revealed that the catechol core is essential for activity and can covalently bind to SARS-CoV-2 3CLpro. Among other 45 catechol derivatives, wedelolactone, capsazepine and brazilin showed better SARS-CoV-2 3CLpro inhibitory activities with IC50 values of 1.35 µM, 1.95 µM and 1.18 µM, respectively. These catechol derivatives were verified to be irreversible covalent inhibitors by time-dependent experiments, enzymatic kinetic studies, dilution and dialysis assays. It also exhibited good selectivity towards different cysteine proteases (SARS-CoV-2 PLpro, cathepsin B and cathepsin L). Subsequently, the binding affinity between brazilin and SARS-CoV-2 3CLpro was determined by SPR assay with KD value of 0.80 µM. Molecular dynamic (MD) simulations study showed the binding mode of brazilin in the target protein. In particular, brazilin displayed good anti-SARS-CoV-2 activity in A549-hACE2-TMPRSS2 cells with EC50 values of 7.85 ± 0.20 µM and 5.24 ± 0.21 µM for full time and post-infection treatments, respectively. This study provides a promising lead compound for the development of novel anti-SARS-CoV-2 drugs.

Single-cell characterization of macrophages in uveal melanoma uncovers transcriptionally heterogeneous subsets conferring poor prognosis and aggressive behavior.

Uveal melanoma (UM) is the most frequent primary intraocular malignancy with high metastatic potential and poor prognosis. Macrophages represent one of the most abundant infiltrating immune cells with diverse functions in cancers. However, the cellular heterogeneity and functional diversity of macrophages in UM remain largely unexplored. In this study, we analyzed 63,264 single-cell transcriptomes from 11 UM patients and identified four transcriptionally distinct macrophage subsets (termed MΦ-C1 to MΦ-C4). Among them, we found that MΦ-C4 exhibited relatively low expression of both M1 and M2 signature genes, loss of inflammatory pathways and antigen presentation, instead demonstrating enhanced signaling for proliferation, mitochondrial functions and metabolism. We quantified the infiltration abundance of MΦ-C4 from single-cell and bulk transcriptomes across five cohorts and found that increased MΦ-C4 infiltration was relevant to aggressive behaviors and may serve as an independent prognostic indicator for poor outcomes. We propose a novel subtyping scheme based on macrophages by integrating the transcriptional signatures of MΦ-C4 and machine learning to stratify patients into MΦ-C4-enriched or MΦ-C4-depleted subtypes. These two subtypes showed significantly different clinical outcomes and were validated through bulk RNA sequencing and immunofluorescence assays in both public multicenter cohorts and our in-house cohort. Following further translational investigation, our findings highlight a potential therapeutic strategy of targeting macrophage subsets to control metastatic disease and consistently improve the outcome of patients with UM.

Reply to Correspondence on "Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1''.

The high-resolution X-ray crystal structure of the ternary complex FtmOx1 ⋅ 2OG ⋅ fumitremorgin B and the catalytic mechanism were recently reported by us (DOI 10.1002/anie.202112063). In their Correspondence, Zhang, Costello, Liu et al. criticize our work in several aspects. Herein, we address these questions one by one. These structural clarifications and new computational results further support the CarC-like mechanistic model.

Nature-inspired catalytic asymmetric rearrangement of cyclopropylcarbinyl cation.

In nature, cyclopropylcarbinyl cation is often involved in cationic cascade reactions catalyzed by natural enzymes to produce a great number of structurally diverse natural substances. However, mimicking this natural process with artificial organic catalysts remains a daunting challenge in synthetic chemistry. We report a small molecule-catalyzed asymmetric rearrangement of cyclopropylcarbinyl cations, leading to a series of chiral homoallylic sulfide products with good to excellent yields and enantioselectivities (up to 99% enantiomeric excess). In the presence of a chiral SPINOL-derived N-triflyl phosphoramide catalyst, the dehydration of prochiral cyclopropylcarbinols occurs rapidly to generate symmetrical cyclopropylcarbinyl cations, which are subsequently trapped by thione-containing nucleophiles. A subgram-scale experiment and multiple downstream transformations of the sulfide products are further pursued to demonstrate the synthetic utility. Notably, a few heteroaromatic sulfone derivatives could serve as "covalent warhead" in the enzymatic inhibition of severe acute respiratory syndrome coronavirus 2 main protease.

Discovery of Novel Drug-like PHGDH Inhibitors to Disrupt Serine Biosynthesis for Cancer Therapy.

Being the rate-limiting enzyme within the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH) is abnormally overexpressed in numerous malignant tumor cells and is a promising target for cancer treatment. Here, we report a series of novel PHGDH inhibitors using a focused compound screening and structural optimization approach. The lead compound D8 displayed good enzymatic inhibitory activity (IC50 = 2.8 ± 0.1 µM), high binding affinity (Kd = 2.33 µM), and sensitivity to the cell lines with the PHGDH gene amplification or overexpression. Furthermore, D8 was proven to restrict the de novo serine synthesis from glucose within MDA-MB-468 cells. X-ray crystallographic analysis, molecular dynamics simulations, and mutagenesis experiments on PHGDH revealed the binding site at D175 inside the NAD+-binding pocket. Finally, D8 exhibited excellent in vivo pharmacokinetic properties (F = 82.0%) and exerted evident antitumor efficacy in the PC9 xenograft mouse model.

Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1.

The 2-oxoglutarate (2OG)-dependent non-heme enzyme FtmOx1 catalyzes the endoperoxide biosynthesis of verruculogen. Although several mechanistic studies have been carried out, the catalytic mechanism of FtmOx1 is not well determined owing to the lack of a reliable complex structure of FtmOx1 with fumitremorgin B. Herein we provide the X-ray crystal structure of the ternary complex FtmOx1⋅2OG⋅fumitremorgin B at a resolution of 1.22 Å. Our structures show that the binding of fumitremorgin B induces significant compression of the active pocket and that Y68 is in close proximity to C26 of the substrate. Further MD simulation and QM/MM calculations support a CarC-like mechanism, in which Y68 acts as the H atom donor for quenching the C26-centered substrate radical. Our results are consistent with all available experimental data and highlight the importance of accurate complex structures in the mechanistic study of enzymatic catalysis.

Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution.

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105-/His224+ rather than a concerted process.

Stereodivergent Protein Engineering of a Lipase To Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters.

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.

"Top" or "bottom" switches of a cyclohexanone monooxygenase controlling the enantioselectivity of the sandwiched substrate.

Single mutation at the switch residues F432 (F432I/L) or L435 (L435A/G) efficiently reversed the inherent enantiopreference of WT CHMOAcineto in the Baeyer-Villiger oxidation of various 4-phenyl-cyclohexanone derivatives and 4-alkyl-cyclohexanones, producing a series of substituted lactones with inversed configuration (up to 99% ee and 99% conversion).

Antitumor gemcitabine conjugated micelles from amphiphilic comb-like random copolymers.

Gemcitabine is an important pyrimidine antimetabolite that inhibits cellular DNA synthesis. However, the therapeutic efficacy and clinical benefit of gemcitabine are severely compromised due to its rapid plasma metabolism and low selectivity towards tumor tissues. To overcome these limitations, we prepared novel PEGylated gemcitabine-contained comb-like copolymers poly(monomethoxyl PEG350 methylacrylate -co- 5'-O-vinyladipyl- gemcitabine) (poly(mPEG350MA-co-VAG) and (poly(mPEG1000MA-co-VAG), which could self-assemble into micelles and displayed enhanced antitumor activity. The copolymers and the formed micelles were well characterized for their structure, critical aggregation concentration (CAC), morphology, cellular uptake, cell cytotoxicity, and controlled drug release. Cellular uptake and in vitro cytotoxicity assays against human lung cancerous cells (A549) demonstrated that these micelles could be effectively internalized and induced cell apoptosis. These micelles efficiently inhibited tumor growth when injected intravenously into A549 cell derived xenograft tumor bearing Balb/C nude mice using a dose of 10mg/kg in terms of reduced tumor volume compared to free gemcitabine. In conclusion, PEGylated micelles could protect gemcitabine from rapid plasma metabolism, provided a sustained release and showed enhanced antitumor activity, thus have the potential to be used as novel anticancer drug delivery vehicle.