The Tumor Proteolytic Landscape: A Challenging Frontier in Cancer Diagnosis and Therapy

In recent decades, dysregulation of proteases and atypical proteolysis have become increasingly recognized as important hallmarks of cancer, driving community-wide efforts to explore the proteolytic landscape of oncologic disease. With more than 100 proteases currently associated with different aspects of cancer development and progression, there is a clear impetus to harness their potential in the context of oncology. Advances in the protease field have yielded technologies enabling sensitive protease detection in various settings, paving the way towards diagnostic profiling of disease-related protease activity patterns. Methods including activity-based probes and substrates, antibodies, and various nanosystems that generate reporter signals, i.e., for PET or MRI, after interaction with the target protease have shown potential for clinical translation. Nevertheless, these technologies are costly, not easily multiplexed, and require advanced imaging technologies. While the current clinical applications of protease-responsive technologies in oncologic settings are still limited, emerging technologies and protease sensors are poised to enable comprehensive exploration of the tumor proteolytic landscape as a diagnostic and therapeutic frontier. This review aims to give an overview of the most relevant classes of proteases as indicators for tumor diagnosis, current approaches to detect and monitor their activity in vivo, and associated therapeutic applications.     

Interrelated Mechanism by Which the Methide Quinone Celastrol,Obtained from the Roots of Tripterygium wilfordii,Inhibits Main Protease 3CLpro of COVID-19 and Acts as Superoxide Radical Scavenger

We describe the potential anti coronavirus disease 2019 (COVID-19) action of the methide quinone inhibitor, celastrol. The related methide quinone dexamethasone is, so far, among COVID-19 medications perhaps the most effective drug for patients with severe symptoms. We observe a parallel redox biology behavior between the antioxidant action of celastrol when scavenging the superoxide radical, and the adduct formation of celastrol with the main COVID-19 protease. The related molecular mechanism is envisioned using molecular mechanics and dynamics calculations. It proposes a covalent bond between the S(Cys145) amino acid thiolate and the celastrol A ring, assisted by proton transfers by His164 and His41 amino acids, and a π interaction from Met49 to the celastrol B ring. Specifically, celastrol possesses two moieties that are able to independently scavenge the superoxide radical: the carboxylic framework located at ring E, and the methide-quinone ring A. The latter captures the superoxide electron, releasing molecular oxygen, and is the feature of interest that correlates with the mechanism of COVID-19 inhibition. This unusual scavenging of the superoxide radical is described using density functional theory (DFT) methods, and is supported experimentally by cyclic voltammetry and X-ray diffraction.     

Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach

Pharmacophore searches that include anchors, fragments contributing above average to receptor binding, combined with one-step syntheses are a powerful approach for the fast discovery of novel bioactive molecules. Here, we are presenting a pipeline for the rapid and efficient discovery of aspartyl protease inhibitors. First, we hypothesized that hydrazine could be a multi-valent warhead to interact with the active site Asp carboxylic acids. We incorporated the hydrazine anchor in a multicomponent reaction and created a large virtual library of hydrazine derivatives synthetically accessible in one-step. Next, we performed anchor-based pharmacophore screening of the libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an enzyme activity assay and the binding mode confirmed by several soaked crystal structures supporting the validity of the hypothesis and approach. The herein reported pipeline of tools will be of general value for the rapid generation of receptor binders beyond Asp proteases.     

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His₆-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.     

Pharmacokinetic Parameters of HIV-1 Protease Inhibitors

Since the beginning of the HIV epidemic, research has been carried out to control the virus. Understanding the mechanisms of replication has given access to the various classes of drugs that over time have transformed AIDS into a manageable chronic disease. The class of protease inhibitors (PIs) gained notice in anti-retroviral therapy, once it was found that peptidomimetic molecules act by blocking the active catalytic center of the aspartic protease, which is directly related to HIV maturation. However, mutations in enzymatic internal residues are the biggest issue for these drugs, because a small change in biochemical interaction can generate resistance. Low plasma concentrations of PIs favor viral natural selection; high concentrations can inhibit even partially resistant enzymes. Food-drug/drug-drug interactions can decrease the bioavailability of PIs and are related to many side effects. Therefore, this review summarizes the pharmacokinetic properties of current PIs, the changes when pharmacological boosters are used and also lists the major mutations to help understanding of how long the continuous treatment can ensure a low viral load in patients.     

Effects of extracellular proteases and its inhibitors on the gel characteristics of soy protein induced by lactic acid bacteria

The purpose of this study was to analyse the relationship between extracellular protease activity and gel characteristics of soybean protein gel induced by lactic acid bacteria (LAB), the influence of protease inhibition on the gel characteristics was discussed through SDS-PAGE, rheological properties and microstructure. The results showed that the pH value of Lactobacillus casei decreased slowly, but the gel could be formed at 2 h, maybe its higher extracellular protease activity improved the gel characteristics. The addition of ethylenediaminetetraacetic acid (10 mmol L⁻¹) reduced the gel quality, when the protease activity was inhibited, the gel quality would decrease, even if there were macromolecular crosslinkers in the system. This study showed that extracellular protease had an important influence on gel characteristics and provided a theoretical basis for the application of protease producing LAB in fermentation-induced soybean gel foods.     

Modified enzymatic collagen digestion-mediated isolation of osteocytes

This study established a method for isolating large numbers of high-purity osteocytes from high-density bone. Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times, and the digested cells and bone chips (BC) were cultured, digested, and passaged when cells were fully grown. The types of cells obtained were identified by morphology, viable cell counts, alkaline phosphatase staining, and biochemical activity analyses, and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction. Our results showed that among the cells obtained from the third digestion (fractions 7–9) of wild mice tibias and femurs and the remaining BCs, 85%–90% of the cells were osteocytes. Moreover, their morphology was approximately one-tenth to one-fifth the size of osteoblasts, star-shaped or polygonal, with a dendritic structure, negative for alkaline phosphatase staining, and showed a high expression of dmp1 and sclerostin. Ninety percent of the cells in fractions 1–3 were osteoblasts, and were fusiform or polygonal shape. The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction, while the expression of osteocyte-specific dmp1 and sclerostin was not detected. In the second portion (fractions 4–6), a large number were osteoblasts, mixed with a small number of osteocytes, and had high alkaline phosphatase activity and osteocyte mRNA levels, a specific level of the osteocyte marker dmp1, and no sclerostin was detected. Osteocytes in daβcat^ot mice were also successfully isolated by this method, and we found that Wnt signaling increased the proliferation of these osteocytes. The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone, which provides an avenue for the study of osteocyte function in vitro.     

猪血蛋白水解液氨基酸成分分析

以新鲜猪血为原料,制得了猪血蛋白干粉,选用三种不同类别的蛋白酶在适宜的条件下水解猪血蛋白干粉,并得到精制水解蛋白液。用氨基酸分析仪检测水解液中氨基酸种类及含量,并与医用脑蛋白液进行比较分析。结果表明胰蛋白酶水解效果最好,水解液成分与医用脑蛋白液成分比较接近。     

碱性蛋白酶洗涤稳定性提高的研究进展

碱性蛋白酶是液体洗涤剂中使用量最大的一类酶制剂,应用于洗涤剂行业的碱性蛋白酶占碱性蛋白酶市场的60%以上。不同于粉状洗涤剂中的酶制剂被造粒所包裹,液体洗涤剂中的碱性蛋白酶直接暴露于溶液中,与洗涤剂成分如表面活性剂、洗涤助剂及漂白剂直接接触作用,使得蛋白酶洗涤稳定性下降。除此之外,蛋白酶在液体洗涤剂环境中存在普遍的自溶失活现象,对其洗涤性能造成不利影响。如何提升碱性蛋白酶的稳定性是液体洗涤剂领域中的一个热点问题。本文介绍了碱性蛋白酶的催化与失活机制,综述了几种常用于稳定碱性蛋白酶的策略,即添加稳定剂、分子改造和化学修饰,重点介绍了化学修饰中的聚乙二醇化修饰与多糖修饰,对比了两种修饰方法的过程与效果,并在最后对碱性蛋白酶稳定性提高策略进行了展望。