A robust computational quest: Discovering potential hits to improve the treatment of pyrazinamide-resistant Mycobacterium tuberculosis.

The rise of pyrazinamide (PZA)-resistant strains of Mycobacterium tuberculosis (MTB) poses a major challenge to conventional tuberculosis (TB) treatments. PZA, a cornerstone of TB therapy, must be activated by the mycobacterial enzyme pyrazinamidase (PZase) to convert its active form, pyrazinoic acid, which targets the ribosomal protein S1. Resistance, often associated with mutations in the RpsA protein, complicates treatment and highlights a critical gap in the understanding of structural dynamics and mechanisms of resistance, particularly in the context of the G97D mutation. This study utilizes a novel integration of computational techniques, including multiscale biomolecular and molecular dynamics simulations, physicochemical and medicinal chemistry predictions, quantum computations and virtual screening from the ZINC and Chembridge databases, to elucidate the resistance mechanism and identify lead compounds that have the potential to improve treatment outcomes for PZA-resistant MTB, namely ZINC15913786, ZINC20735155, Chem10269711, Chem10279789 and Chem10295790. These computational methods offer a cost-effective, rapid alternative to traditional drug trials by bypassing the need for organic subjects while providing highly accurate insight into the binding sites and efficacy of new drug candidates. The need for rapid and appropriate drug development emphasizes the need for robust computational analysis to justify further validation through in vitro and in vivo experiments.

An electrochemical sensor based on molecularly imprinted poly(o-phenylenediamine) for the detection of thymol.

A molecularly imprinted electrochemical sensor was facilely fabricated for the detection of thymol (THY). o-Phenylenediamine (oPD) was used as the functional monomer and electropolymerized on the surface of the glassy carbon electrode (GCE) by using THY as the templates. After the THY templates were removed with 50 % (v/v) ethanol, imprinted cavities complementary to the templates were formed within the poly(o-phenylenediamine) (PoPD) films. The resultant molecularly imprinted PoPD/GCE (MI-PoPD/GCE) was used for the detection of THY, and a wide linear range from 0.5 to 100 µM with a low limit of detection (LOD) of 0.084 µM were obtained under the optimal conditions. The developed MI-PoPD/GCE also displays high selectivity, reproducibility and stability for THY detection. Finally, the content of THY in the real samples was accurately determined by the as-fabricated MI-PoPD/GCE, demonstrating its high practicability and reliability.

Fabrication of Super-Sized Metal Inorganic-Organic Hybrid Glass with Supramolecular Network via Crystallization-Suppressing Approach.

Metal coordination compound (MCC) glasses [e.g., metal-organic framework (MOF) glass, coordination polymer glass, and metal inorganic-organic complex (MIOC) glass] are emerging members of the hybrid glass family. So far, a limited number of crystalline MCCs can be converted into glasses by melt-quenching. Here, we report a universal wet-chemistry method, by which the super-sized supramolecular MIOC glasses can be synthesized from non-meltable MOFs. Alcohol and acid were used as agents to inhibit crystallization. The MIOC glasses demonstrate unique features including high transparency, shaping capability, and anisotropic network. Directional photoluminescence with a large polarization ratio (≈47 %) was observed from samples doped with organic dyes. This crystallization-suppressing approach enables fabrication of super-sized MCC glasses, which cannot be achieved by conventional vitrification methods, and thus allows for exploring new MCC glasses possessing photonic functionalities.

Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides.

Lasso peptides are unique natural products that comprise a class of ribosomally synthesized and post-translationally modified peptides. Their defining three-dimensional structure is a lariat knot, in which the C-terminal tail is threaded through a macrolactam ring formed between the N-terminal amino group and an Asp or Glu side chain (i.e., an isopeptide bond). Recent genome mining strategies have revealed various types of lasso peptide biosynthetic gene clusters and have thus redefined the known chemical space of lasso peptides. To date, over 20 different types of these gene clusters have been discovered, including several different clades from Proteobacteria. Despite the diverse architectures of these gene clusters, which may or may not encode various tailoring enzymes, most currently known lasso peptides are synthesized by two discrete clades defined by the presence of an ATP-binding cassette transporter or its absence and (sometimes) concurrent appearance of an isopeptidase, raising questions about their evolutionary history. Herein, we discovered and characterized the lasso peptide rubrinodin, which is assembled by a gene cluster encoding both an ATP-binding cassette transporter and an isopeptidase. Our bioinformatics analyses of this and other representative cluster types provided new clues into the evolutionary history of lasso peptides. Furthermore, our structural and biochemical investigations of rubrinodin permitted the conversion of this thermolabile lasso peptide into a more thermostable scaffold.

SARS-CoV-2 omicron variant clearance delayed in breakthrough cases with elevated fasting blood glucose.

BACKGROUND: Omicron variant (B.1.1.529) is a dominant variant worldwide. However, the risk factors for Omicron variant clearance are yet unknown. The present study aimed to investigate the risk factors for early viral clearance of Omicron variant in patients with a history of inactivated vaccine injection. METHODS: Demographic, clinical, and epidemiological data from 187 patients were collected retrospectively during the Omicron variant wave. RESULTS: 73/187 and 114/187 patients were administered two and three doses of vaccine, respectively. The median duration of SARS-CoV-2 RNA positivity was 9 days, and the difference between patients with two and three vaccine injections was insignificant (P = 0.722). Fever was the most common symptom (125/187), and most patients (98.4%) had a fever for < 7 days. The RNA was undetectable in 65/187 patients on day 7. Univariable logistic analysis showed that baseline glucose, uric acid, lymphocytes count, platelet count, and CD4+ T lymphocyte count were associated with SARS-CoV-2 RNA-positivity on day 7. Multivariable analysis showed that glucose ≥ 6.1 mmol/L and CD4+T lymphocytes count were independent risk factors for RNA positivity on day 7. 163/187 patients had an undetectable RNA test on day 14, and uric acid was the only independent risk factor for RNA positivity. Moreover, baseline glucose was negatively correlated with uric acid and CD4+ and CD8+ T cell count, while uric acid was positively correlated with CD4+ and CD8+ T cell count. CONCLUSIONS: Omicron variant clearance was delayed in breakthrough cases with elevated fasting blood glucose, irrespective of the doses of inactivated vaccine.

Design and synthesis of cinnamic acid triptolide ester derivatives as potent antitumor agents and their biological evaluation.

A series of novel cinnamic acid triptolide ester derivatives were synthesized, and their growth inhibitory properties against human hepatoma HepG2 cells were assessed as the measure of cytotoxicity with triptolide as the positive control. One of the phenolic hydroxyl phosphorylated products, CL20 was found to possess the best cytotoxicity and surpassed the parent natural triptolide, suggesting that compound CL20 is a promising antitumor lead compound and deserves further research of pharmacological activity. In addition, the structure-activity relationship for these compounds was also investigated.

Design, synthesis, and bioactivity evaluation of macrocyclic benzo[b]pyrido[4,3-e][1,4]oxazine derivatives as novel Pim-1 kinase inhibitors.

Pim-1 kinase is a serine/threonine kinase which is vital in many tumors. The Pim-1 inhibitor 10-DEBC and its derivatives discovered in our previous work were modified through macrocyclization strategy. A series of benzo[b]pyridine[4,3-e][1,4]oxazine macrocyclic compounds were designed, synthesized, and evaluated as novel Pim-1 kinase inhibitors. Among these compounds, compound H5 exhibited the highest activity with an IC50 value of 35 nM. In addition, the crystal complex structure of Pim-1 kinase bound with compound H3 was determined, and the structure-activity relationship of these macrocyclic compounds was analyzed, which provides the structural basis of further optimization of novel macrocyclic Pim-1 kinase inhibitors..

Construction and validation of a predictive model for hepatocellular carcinoma based on serum markers.

BACKGROUND: Early hepatocellular carcinoma (HCC) detection with non-invasive biomarkers remains an unmet clinical need. We aimed to construct a predictive model based on the pre-diagnostic levels of serum markers to predict the early-stage onset of HCC. METHODS: A total of 339 HCC patients (including 157 patients from Changzhou cohort and 182 patients from Wuxi cohort) were enrolled in our retrospective study. Levels of 25 baseline serum markers were collected. Propensity score matching (PSM) analysis was conducted to balance the distributions of patients' gender, age, and the surveillance time between HCC group and control group. Then, Receiver operating characteristic (ROC) and Logistic regression analysis were performed to screen the independent predictive variables and construct a non-invasive predictive model. Subsequently, ROC curve and Kaplan-Meier (K-M) curve were used to evaluate the predictive values of the model. Clinical net benefit of the model was demonstrated by decision curve analysis (DCA) and clinical impact curve. RESULTS: Five independent predictive variables for HCC onset and two general characteristics of patients (age and gender) were incorporated into the score model. ROC and DCA curves showed that the score model had better predictive performance in discrimination and clinical net benefit compared with single variable or other score systems, with the area under the curve (AUC) of 0.890 (95% CI 0.856-0.925) in Changzhou cohort and 0.799 (95% CI 0.751-0.849) in Wuxi cohort. Meanwhile, stratification analysis indicated that the score model had good predictive values for patients with early tumor stage (AJCC stage I) or small tumors (< 2 cm). Moreover, the score of HCC patient began to increase at 30 months before clinical diagnosis and reach a peak at 6 months. CONCLUSION: Based on this model, we could optimize the current risk stratification at an early stage and consider further intensive surveillance programs for high-risk patients. It could also help clinicians to evaluate the progression and predict the prognosis of HCC patients.

Near-infrared laser driven white light continuum generation: materials, photophysical behaviours and applications.

The pursuit of efficient light sources has stimulated continued effort in the search of materials and methods for generating white light emission. In addition to the white light produced by light-emitting diodes (LEDs) and fluorescent lamps that involves spectral conversion of high energy to low energy emission, recent studies showed that it was also possible to produce white visible light by irradiating different active materials with near-infrared (NIR) constant-wave (CW) lasers. In this review, we begin by introducing and categorizing different materials that exhibit NIR laser driven white light emission, including normal inorganic phosphors, organometallic compounds, graphene, etc. We then discuss the photophysical behavior of this process in terms of optical spectra, temperature evolution and photoelectric response. Different mechanisms of while light generation are analyzed afterwards, and the possibility of a more general physical picture of this process is discussed. This review is concluded with a summary of the current understanding and discussion on potential applications and future perspectives.

Structure-Based Optimization of 10-DEBC Derivatives as Potent and Selective Pim-1 Kinase Inhibitors.

Pim-1 kinase has been widely regarded as an attractive target for anticancer drugs. Here, we reported our continued efforts in structure-based optimization of compound 10-DEBC, a previously identified micromolar Pim-1 inhibitor. Guided by the Site Identification by Ligand Competitive Saturation (SILCS) method, we quickly obtained a series of 10-DEBC derivatives with significantly improved activity and selectivity. In particular, compound 26 exhibited an IC50 value of 0.9 nM, as well as 220- and 8-fold selectivity over Pim-2 and Pim-3 kinases, respectively.

Genome mining integrating semi-rational protein engineering and nanoreactor design: roadmap for a robust biocatalyst for industrial resolution of Vince lactam.

Biomanufacturing of chemicals using biocatalysts is an attractive strategy for the production of valuable pharmaceuticals since it is usually more economical and has a much-reduced environmental impact. However, there are often challenges such as their thermal instability that should be overcome before a newly discovered enzyme is eventually translated into industrial processes. In this work, we describe a roadmap for the development of a robust catalyst for industrial resolution of Vince lactam, a key intermediate for the synthesis of carbocyclic-nucleoside-related pharmaceuticals. By a genome mining strategy, a new (+)-γ-lactamase (MiteL) from Microbacterium testaceum was successfully discovered and biochemically characterized. In vitro studies showed that the enzyme exhibited high activity but poor enantioselectivity (E = 6.3 ± 0.2) toward racemic Vince lactam, and thus, it is not suitable for industrial applications. Based on structural modeling and docking studies, a semi-rational engineering strategy combined with an efficient screening method was then applied to improve the enantioselectivity of MiteL. Several mutants with significant shifting stereoselectivity toward (-)-γ-lactam were obtained by site-saturation mutagenesis. Synergy effects led to the final mutant F14D/Q114R/M117L, which enabled efficient acquisition of (-)-γ-lactam with a high E value (> 200). The mutant was biochemically characterized, and the docking studies suggested a plausible mechanism for its improved selectivity. Finally, a sunflower-like nanoreactor was successfully constructed to improve the mutant's robustness via protein supramolecular self-assembly. Thus, the synergism between semi-rational protein engineering and self-assembling immobilization enabled construction of a nanoreactor with superior properties, which can be used for resolution of Vince lactam in large scale.

New perspectives on the treatment of mycobacterial infections using antibiotics.

More than 100 years have passed since the discovery of Mycobacterium tuberculosis, in 1882, as the pathogen that causes tuberculosis (TB). However, globally, TB is still one of the leading causes of death by infectious diseases. In 2018, approximately 10.0 million people were diagnosed with TB owing to the development of advanced strategies by M. tuberculosis to resist antibiotics, including the development of a dormant state. The World Health Organization (WHO) and the Sustainable Development Goals (SDGs) are dedicated to ending TB by 2030. However, the development of strategies to discover new TB drugs and new therapies is crucial for the achievement of this goal. Unfortunately, the rapid occurrence of multidrug-resistant strains of M. tuberculosis has worsened the current situation, thereby warranting prioritized discovery of new anti-TB drugs and the development of new treatment regimens in academia and the pharmaceutical industry. In this mini review, we provide a brief overview of the current research and development pipeline for new anti-TB drugs and present our perspective of TB drug innovation. The data presented herein may enable the introduction of more effective medicines and therapeutic regimens into the market.Key Points• The Updated Global New TB Drug Pipelines are briefly summarized.• Novel strategies for the discovery of new TB drugs, including novel sources, bioinformatics, and synthetic biology strategies, are discussed.• New therapeutic options, including living therapeutics and phage therapy, are proposed.

Enantioselective synthesis of a chiral intermediate of himbacine analogs by Burkholderia cepacia lipase A.

The enantiomers of (4R/S)-4-hydroxy-N, N-diphenyl-2-pentynamide are key chiral synthons for the synthesis of thrombin receptor antagonists such as vorapaxar. In this paper, we report the enzymatic preparation of enantiomerically enriched (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide using lipase A from Burkholderia cepacia ATCC 25416 as the catalyst. First, the lipase gene (lipA) and its chaperone gene (lipB) was cloned and expressed in Escherichia coli system. After purification, lipase A activation was performed with the assistance of foldase lipase B. Enzyme assay revealed that the activated lipase A showed the optimal catalytic activity at 60 ºC and pH 7. The effects of various metals on the activity were investigated and results demonstrated that most of the metals inhibited the activity. To further improve the catalytic outcome, two-phase reaction was studied, and n-hexane proved to be a good organic solvent for the combination system. Using the optimize conditions, (4R)-4-hydroxy-N, N-diphenyl-2-pentynamide with 94.5% ee value and 48.93% conversion ratio was achieved. Our investigation on this lipase reveals lipase A as a promising biocatalyst for producing chiral propargyl alcohol for preparation of novel himbacine analogs.

Discovery of Novel Pim-1 Kinase Inhibitors with a Flexible-Receptor Docking Protocol.

A flexible-receptor docking protocol was designed for treating binding-site side-chain flexibility by integrating essential aspects of "Conformational Selection" and "Induced Fit" in a hierarchical fashion. Assessed in a diverse set of pharmaceutically relevant targets, this protocol showed improved performance in reproducing binding poses and ligand enrichment studies compared to rigid-receptor docking. Moreover, it has also exhibited encouraging efficiency in prospective ligand discovery for Pim-1 kinase, which led to novel Pim-1 inhibitors with single-digit nanomolar potencies.

Enhancement in the catalytic activity of Sulfolobus solfataricus P2 (+)-γ-lactamase by semi-rational design with the aid of a newly established high-throughput screening method.

(-)-γ-Lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one) has attracted increasing attention as the chiral intermediate of carbocyclic nucleosides most of which serve as pharmaceutical agents such as anti-HIV/HBV drugs abacavir and carbovir. So far, developing in vitro (+)-γ-lactamase-mediated biotransformation has been one of the most efficient approaches for the production of (-)-γ-lactam. In this study, the catalytic activity of the (+)-γ-lactamase from Sulfolobus solfataricus P2 was engineered by semi-rational design. Molecular docking and molecular dynamics simulation were carried out to target the key positions relevant to catalytic activity. Nine amino acid residues were selected for site saturation mutagenesis. To expedite the screening process, a sensitive colorimetric high-throughput screening method was established based on the Rimini test which was originally applied to distinguish primary amines from secondary amines. The screening process resulted in the achievement of several efficient mutants: V203N, V203Q, I336H, I336R, and Y388H. Synergy effects led to four final mutants (V203N/I336R, V203N/Y388H, I336R/Y388H, and V203N/I336R/Y388H) with enhanced enzyme activity after the combination of positive single mutants. The best mutant V203N/Y388H/I336R displayed a 21-fold higher enzyme efficiency (kcat/KM) compared to the wild-type enzyme. The result demonstrated that the biotransformation using the triple mutant as the catalyst reached > 49% conversion and > 99% enantiomeric excess at 80 °C after 2 h, which made it a good catalyst candidate to produce (-)-γ-lactam. The possible mechanism responsible for the improvement in the catalytic activity was explicated by analyzing the protein-ligand binding modes and interaction between the protein and the ligand.

Lassomycin and lariatin lasso peptides as suitable antibiotics for combating mycobacterial infections: current state of biosynthesis and perspectives for production.

Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward Mycobacterium tuberculosis, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.

Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway.

Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.

Nanoreactor Design Based on Self-Assembling Protein Nanocages.

Self-assembling proteins that form diverse architectures are widely used in material science and nanobiotechnology. One class belongs to protein nanocages, which are compartments with nanosized internal spaces. Because of the precise nanoscale structures, proteinaceous compartments are ideal materials for use as general platforms to create distinct microenvironments within confined cellular environments. This spatial organization strategy brings several advantages including the protection of catalyst cargo, faster turnover rates, and avoiding side reactions. Inspired by diverse molecular machines in nature, bioengineers have developed a variety of self-assembling supramolecular protein cages for use as biosynthetic nanoreactors that mimic natural systems. In this mini-review, we summarize current progress and ongoing efforts creating self-assembling protein based nanoreactors and their use in biocatalysis and synthetic biology. We also highlight the prospects for future research on these versatile nanomaterials.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one).

To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (-)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (-)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam.IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (-)-Vince lactam.

Construction of an organelle-like nanodevice via supramolecular self-assembly for robust biocatalysts.

BACKGROUND: When using the microbial cell factories for green manufacturing, several important issues need to be addressed such as how to maintain the stability of biocatalysts used in the bioprocess and how to improve the synthetic efficiency of the biological system. One strategy widely used during natural evolution is the creation of organelles which can be used for regional control. This kind of compartmentalization strategy has inspired the design of artificial organelle-like nanodevice for synthetic biology and "green chemistry". RESULTS: Mimicking the natural concept of functional compartments, here we show that the engineered thermostable ketohydroxyglutarate aldolase from Thermotoga maritima could be developed as a general platform for nanoreactor design via supramolecular self-assembly. An industrial biocatalyst-(+)-γ-lactamase was selected as a model catalyst and successful encapsulated in the nanoreactor with high copies. These nanomaterials could easily be synthesized by Escherichia coli by heterologous expression and subsequently self-assembles into the target organelle-like nanoreactors both in vivo and in vitro. By probing their structural characteristics via transmission electronic microscopy and their catalytic activity under diverse conditions, we proved that these nanoreactors could confer a significant benefit to the cargo proteins. The encapsulated protein exhibits significantly improved stability under conditions such as in the presence of organic solvent or proteases, and shows better substrate tolerance than free enzyme. CONCLUSIONS: Our biodesign strategy provides new methods to develop new catalytically active protein-nanoreactors and could easily be applied into other biocatalysts. These artificial organelles could have widely application in sustainable catalysis, synthetic biology and could significantly improve the performance of microbial cell factories.