Efficacy of nature killer cell combination chemotherapy for post-radical gastric cancer metastases: Case report.

Nature killer cell therapy has shown strong efficacy in the field of oncology in recent years and has been applied to patients with metastases with the aim of improving the prognosis of advanced gastric cancer. A 59-year-old male with gastric adenocarcinoma with pancreatic metastasis (T4N0M1) who underwent radical surgery for gastric cancer with tumor metastasis was treated with oxaliplatin and tegafur combined with cellular reinfusion in stages. Computed tomograpy scan and serum tumor markers were monitored continuously after the treatment course. After five courses of combined treatment, the patient was in disease control with no significant side effects. At the last follow-up, the alpha fetoprotein had returned to its normal value with a poor display of low-density shadows in the body of the pancreas. Pancreatic cancer is insidious in origin and has a high mortality rate. The report provides clinical evidence for cell therapy of pancreatic metastatic cancer with improved quality of life.

Computation-Guided Rational Design of Cysteine-Less Protein Variants in Engineered hCGL.

The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated Tm value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.

Computational design of α-amylase from Bacillus licheniformis to increase its activity and stability at high temperatures.

The thermostable α-amylase derived from Bacillus licheniformis (BLA) has multiple advantages, including enhancing the mass transfer rate and by reducing microbial contamination in starch hydrolysis. Nonetheless, the application of BLA is constrained by the accessibility and stability of enzymes capable of achieving high conversion rates at elevated temperatures. Moreover, the thermotolerance of BLA requires further enhancement. Here, we developed a computational strategy for constructing small and smart mutant libraries to identify variants with enhanced thermostability. Initially, molecular dynamics (MD) simulations were employed to identify the regions with high flexibility. Subsequently, FoldX, a computational design predictor, was used to design mutants by rigidifying highly flexible residues, whereas the simultaneous decrease in folding free energy assisted in improving thermostability. Through the utilization of MD and FoldX, residues K251, T277, N278, K319, and E336, situated at a distance of 5 Å from the catalytic triad, were chosen for mutation. Seventeen mutants were identified and characterized by evaluating enzymatic characteristics and kinetic parameters. The catalytic efficiency of the E271L/N278K mutant reached 184.1 g L-1 s-1, which is 1.88-fold larger than the corresponding value determined for the WT. Furthermore, the most thermostable mutant, E336S, exhibited a 1.43-fold improvement in half-life at 95 â„ƒ, compared with that of the WT. This study, by combining computational simulation with experimental verification, establishes that potential sites can be computationally predicted to increase the activity and stability of BLA and thus provide a possible strategy by which to guide protein design.

Roles of the N-terminal motif in improving the activity and soluble expression of phenylalanine ammonia lyases in Escherichia coli.

Phenylalanine ammonia-lyase (PAL) has various applications in fine chemical manufacturing and the pharmaceutical industry. In particular, PAL derived from Anabaena variabilis (AvPAL) is used as a therapeutic agent to the treat phenylketonuria in clinical settings. In this study, we aligned the amino acid sequences of AvPAL and PAL derived from Nostoc punctiforme (NpPAL) to obtain several mutants with enhanced activity, expression yield, and thermal stability via amino acid substitution and saturation mutagenesis at the N-terminal position. Enzyme kinetic experiments revealed that the kcat values of NpPAL-N2K, NpPAL-I3T, and NpPAL-T4L mutants were increased to 3.2-, 2.8-, and 3.3-fold that of the wild-type, respectively. Saturation mutagenesis of the fourth amino acid in AvPAL revealed that the kcat values of AvPAL-L4N, AvPAL-L4P, AvPAL-L4Q and AvPAL-L4S increased to 4.0-, 3.7-, 3.6-, and 3.2-fold, respectively. Additionally, the soluble protein yield of AvPAL-L4K increased to approximately 14 mg/L, which is approximately 3.5-fold that of AvPAL. Molecular dynamics studies further revealed that maintaining the attacking state of the reaction and N-terminal structure increased the rate of catalytic reaction and improved the solubility of proteins. These findings provide new insights for the rational design of PAL in the future.

Identification of potential inhibitors of omicron variant of SARS-Cov-2 RBD based virtual screening, MD simulation, and DFT.

Emergence of the SARS-CoV-2 Omicron variant of concern (VOC; B.1.1.529) resulted in a new peak of the COVID-19 pandemic, which called for development of effective therapeutics against the Omicron VOC. The receptor binding domain (RBD) of the spike protein, which is responsible for recognition and binding of the human ACE2 receptor protein, is a potential drug target. Mutations in receptor binding domain of the S-protein have been postulated to enhance the binding strength of the Omicron VOC to host proteins. In this study, bioinformatic analyses were performed to screen for potential therapeutic compounds targeting the omicron VOC. A total of 92,699 compounds were screened from different libraries based on receptor binding domain of the S-protein via docking and binding free energy analysis, yielding the top 5 best hits. Dynamic simulation trajectory analysis and binding free energy decomposition were used to determine the inhibitory mechanism of candidate molecules by focusing on their interactions with recognized residues on receptor binding domain. The ADMET prediction and DFT calculations were conducted to determine the pharmacokinetic parameters and precise chemical properties of the identified molecules. The molecular properties of the identified molecules and their ability to interfere with recognition of the human ACE2 receptors by receptor binding domain suggest that they are potential therapeutic agents for SARS-CoV-2 Omicron VOC.

Enhanced Rishirilide Biosynthesis by a Rare In-Cluster Phosphopantetheinyl Transferase in Streptomyces xanthophaeus.

Phosphopantetheinyl transferases (PPTases) play important roles in activating apo-acyl carrier proteins (apo-ACPs) and apo-peptidyl carrier proteins (apo-PCPs) in both primary and secondary metabolism. PPTases catalyze the posttranslational modifications of those carrier proteins by covalent attachment of the 4'-phosphopantetheine group to a conserved serine residue. The protein-protein interactions between a PPTase and a cognate acyl or peptidyl carrier protein have important regulatory functions in microbial biosynthesis, but the molecular mechanism underlying their specific recognition remains elusive. In this study, we identified a new rishirilide biosynthetic gene cluster with a rare in-cluster PPTase from Streptomyces xanthophaeus no2. The function of this Sfp-type PPTase, SxrX, in rishirilide production was confirmed using genetic mutagenesis and biochemical characterization. We applied molecular modeling and site-directed mutagenesis to identify key residues mediating the protein-protein interaction between SxrX and its cognate ACP. In addition, six natural products were isolated from wild-type S. xanthophaeus no2 and the ΔsxrX mutant, including rishirilide A and lupinacidin A, that exhibited antimicrobial and anticancer activities, respectively. SxrX is the first Sfp-type PPTase identified from an aromatic polyketide biosynthetic gene cluster and shown to be responsible for high-level production of rishirilide derivatives. IMPORTANCE Genome mining has been a vital means for natural product drug discovery in the postgenomic era. The rishirilide-type polyketides have attracted attention due to their potent bioactivity, but the poor robustness of production hosts has limited further research and development. This study not only identifies a hyperproducer of rishirilides but also reveals a rare, in-cluster PPTase SxrX that plays an important role in boosting rishirilide biosynthesis. Experimental and computational investigations revealed new insights on the protein-protein interaction between SxrX and its cognate ACP with wide implications for understanding polyketide biosynthesis.

The antitumor activity of hPRDX5 against pancreatic cancer and the possible mechanisms.

Recombinant human peroxiredoxin-5 (hPRDX5), isolated from anti-cancer bioactive peptide (ACBPs), shows a homology of 89% with goat peroxiredoxin-5 (gPRDX5) and is reported to display anti-tumor activity in vivo. Herein, we explored the effect of hPRDX5 and the responsible mechanism in treating pancreatic cancer. Tumor-bearing mice were randomly divided into normal PBS group and treatment group (n=5; 10 mg/kg hPRDX5). Flow cytometry was employed to examine lymphocytes, myeloid-derived suppressor cell subsets, and the function proteins of natural killer (NK) cells in peripheral blood, spleen, and tumor tissues of mice. Western blot was used to measure the protein expressions of the key nodes in TLR4-MAPK-NF-κB signaling pathway. The rate of tumor suppression was 57.6% at a 10 mg/kg dose in orthotopic transplanted tumor mice. Moreover, the population of CD3+CD4+T cells, NK cells, and CD3+CD8+T cells was significantly increased in the tumor tissue of the hPRDX5 group, while the proportion of granulocytic-myeloid-derived suppressor cells decreased slightly. In addition, after treatment with hPRDX5, the percentage of NK cells in blood increased more than 4-fold. Our findings indicated that hPRDX5 effectively suppressed pancreatic cancer possibly via the TLR4-MAPK-NF-κB signaling cascade; hence hPRDX5 could be a prospective immunotherapy candidate for treating pancreatic cancer.

Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening.

Directed evolution is a powerful approach to engineer enzymes via iterative creation and screening of variant libraries. However, assay development for high-throughput mutant screening remains challenging, particularly for new catalytic activities. Mass spectrometry (MS) analysis is label-free and well suited for untargeted discovery of new enzyme products but is traditionally limited by slow speed. Here we report an automated workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening. For a proof of concept, we chose to engineer a cyclodipeptide synthase (CDPS) that synthesizes diketopiperazine (DKP) compounds with therapeutic potential. In recombinant Escherichia coli, site-saturation mutagenesis (SSM) and error-prone PCR (epPCR) libraries expressing CDPS mutants were automatically created and cultivated on an integrated work cell. Culture supernatants were then robotically processed for matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS analysis at a rate of 5 s per sample. The resulting mass spectral data were processed via custom computational algorithms, which performed a multivariant analysis of 108 theoretical mass-to-charge (m/z) values of 190 possible DKP molecules within a mass window of 115-373 Da. An F186L CDPS mutant was isolated to produce cyclo(l-Phe-l-Val), which is undetectable in the product profile of the wild-type enzyme. This robotic, label-free MS screening approach may be generally applicable to engineering other enzymes with new activities in high throughput.

Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin.

The potential antimicrobial compound Chuangxinmycin (CXM) targets the tryptophanyl-tRNA synthetase (TrpRS) of both Gram-negative and Gram-positive bacteria. However, the specific steric recognition mode and interaction mechanism between CXM and TrpRS is unclear. Here, we studied this interaction using recombinant GsTrpRS from Geobacillus stearothermophilus by X-ray crystallography and molecular dynamics (MD) simulations. The crystal structure of the recombinant GsTrpRS in complex with CXM was experimentally determined to a resolution at 2.06 Å. After analysis using a complex-structure probe, MD simulations, and site-directed mutation verification through isothermal titration calorimetry, the interaction between CXM and GsTrpRS was determined to involve the key residues M129, D132, I133, and V141 of GsTrpRS. We further evaluated binding affinities between GsTrpRS WT/mutants and CXM; GsTrpRS was found to bind CXM through hydrogen bonds with D132 and hydrophobic interactions between the lipophilic tricyclic ring of CXM and M129, I133, and V141 in the substrate-binding pockets. This study elucidates the precise interaction mechanism between CXM and its target GsTrpRS at the molecular level and provides a theoretical foundation and guidance for the screening and rational design of more effective CXM analogs against both Gram-negative and Gram-positive bacteria.

The antitumor activity of hPRDX5 against pancreatic cancer and the possible mechanisms

Recombinant human peroxiredoxin-5 (hPRDX5), isolated from anti-cancer bioactive peptide (ACBPs), shows a homology of 89% with goat peroxiredoxin-5 (gPRDX5) and is reported to display anti-tumor activity in vivo. Herein, we explored the effect of hPRDX5 and the responsible mechanism in treating pancreatic cancer. Tumor-bearing mice were randomly divided into normal PBS group and treatment group (n=5; 10 mg/kg hPRDX5). Flow cytometry was employed to examine lymphocytes, myeloid-derived suppressor cell subsets, and the function proteins of natural killer (NK) cells in peripheral blood, spleen, and tumor tissues of mice. Western blot was used to measure the protein expressions of the key nodes in TLR4-MAPK-NF-κB signaling pathway. The rate of tumor suppression was 57.6% at a 10 mg/kg dose in orthotopic transplanted tumor mice. Moreover, the population of CD3+CD4+T cells, NK cells, and CD3+CD8+T cells was significantly increased in the tumor tissue of the hPRDX5 group, while the proportion of granulocytic-myeloid-derived suppressor cells decreased slightly. In addition, after treatment with hPRDX5, the percentage of NK cells in blood increased more than 4-fold. Our findings indicated that hPRDX5 effectively suppressed pancreatic cancer possibly via the TLR4-MAPK-NF-κB signaling cascade; hence hPRDX5 could be a prospective immunotherapy candidate for treating pancreatic cancer.

Expression, purification and X-ray crystal diffraction analysis of alcohol dehydrogenase 1 from Artemisia annua L.

Alcohol dehydrogenase 1 identified from Artemisia annua (AaADH1) is a 40 kDa protein that predominately expressed in young leaves and buds, and catalyzes dehydrogenation of artemisinic alcohol to artemisinic aldehyde in artemisinin biosynthetic pathway. In this study, AaADH1 encoding gene was subcloned into vector pET-21a(+) and expressed in Escherichia coli. BL21(DE3), and purified by Co2+ affinity chromatography. Anion exchange chromatography was performed until the protein purity reached more than 90%. Crystallization of AaADH1 was conducted for further investigation of the molecular mechanism of catalysis, and hanging-drop vapour diffusion method was used in experiments. The results showed that the apo AaADH1 crystal diffracted to 2.95 Å resolution, and belongs to space group P1, with unit-cell parameters, a = 77.53 Å, b = 78.49 Å, c = 102.44 Å, α = 71.88°, ß = 74.02°, γ = 59.97°. The crystallization condition consists of 0.1 M Bis-Tris pH 6.0, 13% (w/v) PEG 8000 and 5% (v/v) glycerol.

Discovery of Antitumor Active Peptides Derived from Peroxiredoxin 5.

The peroxiredoxin 5 (PRDX5) is a member of peroxiredoxins with antitumor activity. However, as a recombinant protein, PRDX5 is restricted in clinic due to high cost and keeping high dose in medication. The alternative way is to explore the antitumor active fragments of PRDX5 for potential of peptide drugs. According to the sequence, crystal structure and enzyme function of PRDX5, seven peptides were designed and named as IMB-P1∼7. The peptide IMB-P1 (AFTPGCSKTHLPGFVEQAEAL) containing critical residue C47 exhibited antitumor activity similar to PRDX5 in vivo. Transcriptome analysis showed peptide IMB-P1 could make influence on expression of multiple genes involved in tumorigenesis and deterioration. Besides, an important discovery is the down-regulation of oxidation-related genes. In CT26 cells, IMB-P1 carried similar antitumor activity with increasing ROS level to intact PRDX5. The results demonstrated that peptide IMB-P1 with easier synthesis from PRDX5 may serve as a promising antitumor candidate.

The Complex Structure of Protein AaLpxC from Aquifex aeolicus with ACHN-975 Molecule Suggests an Inhibitory Mechanism at Atomic-Level against Gram-Negative Bacteria.

New drugs with novel antibacterial targets for Gram-negative bacterial pathogens are desperately needed. The protein LpxC is a vital enzyme for the biosynthesis of lipid A, an outer membrane component of Gram-negative bacterial pathogens. The ACHN-975 molecule has high enzymatic inhibitory capacity against the infectious diseases, which are caused by multidrug-resistant bacteria, but clinical research was halted because of its inflammatory response in previous studies. In this work, the structure of the recombinant UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase from Aquifex aeolicus in complex with ACHN-975 was determined to a resolution at 1.21 Å. According to the solved complex structure, ACHN-975 was docked into the AaLpxC's active site, which occupied the site of AaLpxC substrate. Hydroxamate group of ACHN-975 forms five-valenced coordination with resides His74, His226, Asp230, and the long chain part of ACHN-975 containing the rigid alkynyl groups docked in further to interact with the hydrophobic area of AaLpxC. We employed isothermal titration calorimetry for the measurement of affinity between AaLpxC mutants and ACHN-975, and the results manifest the key residues (His74, Thr179, Tyr212, His226, Asp230 and His253) for interaction. The determined AaLpxC crystal structure in complex with ACHN-975 is expected to serve as a guidance and basis for the design and optimization of molecular structures of ACHN-975 analogues to develop novel drug candidates against Gram-negative bacteria.

NK cells adjuvant therapy shows survival benefits in a gastric mixed signet ring cell carcinoma patient: A case report.

RATIONALE: Advanced signet ring cell (SRC) carcinoma has a worse prognosis. Therefore, early diagnosis and prevention is particularly important; SRC tumors have lower R0 resection rate and are thought to be less chemosensitive than non-SRCC. Consequently, a novel postoperative adjuvant treatment is urgently needed to improve clinical outcomes. PATIENT CONCERNS: A 41-year-old female with advanced gastric SRC carcinoma was treated with radical gastrectomy and oxaliplatin-based regimen for 6 cycles after surgery. She was suspected of recurrence with the high level of carbohydrate antigen (CA) 72-4. DIAGNOSES: The gastroscopy revealed SRC carcinoma of gastric antrum and poorly differentiated adenocarcinoma in some areas. The diagnosis of postoperative pathology report was gastric cancer with stage III C (T4a, N3a, M0). INTERVENTIONS: The level of CA72-4 rapidly increased during the 2 follow-up after the completion of conventional treatment, ex vivo-cultured allogeneic natural killer (NK) cell infusion was offered to prevent recurrence. OUTCOMES: Intravenous injections of NK cells combination with surgical treatment and chemotherapy showed therapeutic effects in this patient with possible relapse. The patient remained disease-free 46 months after the infusion of NK cells until the latest follow-up. LESSONS: CA72-4 appeared to be the most sensitive and specific marker in the gastric cancer patient, and the high level of CA72-4 may indicate the risk of recurrence. This case report provide rationale for NK cell infusion following the rapid increase of CA72-4 to prevent recurrence.

LXYL-P1-2 immobilized on magnetic nanoparticles and its potential application in paclitaxel production

BACKGROUND: LXYL-P1-2 is the first reported glycoside hydrolase that can catalyze the transformation of 7-b-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT) by removing the D-xylosyl group at the C7 position. Successful synthesis of paclitaxel by one-pot method combining the LXYL-P1-2 and 10- deacetylbaccatin III-10-b-O-acetyltransferase (DBAT) using XDT as a precursor, making LXYL-P1-2 a highly promising enzyme for the industrial production of paclitaxel. The aim of this study was to investigate the catalytic potential of LXYL-P1-2 stabilized on magnetic nanoparticles, the surface of which was modified by Ni2+-immobilized cross-linked Fe3O4@Histidine. RESULTS: The diameter of matrix was 20­40 nm. The Km value of the immobilized LXYL-P1-2 catalyzing XDT (0.145 mM) was lower than that of the free enzyme (0.452 mM), and the kcat/Km value of immobilized enzyme (12.952 mM s 1 ) was higher than the free form (8.622 mM s 1 ). The immobilized form maintained 50% of its original activity after 15 cycles of reuse. In addition, the stability of immobilized LXYL-P1-2, maintained 84.67% of its initial activity, improved in comparison with free form after 30 d storage at 4 C. CONCLUSIONS: This investigation not only provides an effective procedure for biocatalytic production of DT, but also gives an insight into the application of magnetic material immobilization technology.

Chemoradiotherapy combined with NK cell transfer in a patient with recurrent and metastatic nasopharyngeal carcinoma inducing long-term tumor control: A case report.

RATIONALE: Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in Southern China. Although combined chemotherapy with radiotherapy has been widely used in treating locally advanced lesions, relapse and metastases remain the primary cause of treatment failure, and are associated with an extremely poor prognosis. Therefore, more efficient and milder therapies are needed. PATIENT CONCERNS: Herein, we report a patient with advanced NPC with intracranial metastases who showed progression during conventional treatment. DIAGNOSES: Nonkeratinizing undifferentiated nasopharyngeal carcinoma (stage IV). INTERVENTIONS: After the completion of initial chemoradiotherapy and targeted therapy, metastases to brain occurred during follow-up. Ex vivo-cultured allogeneic NK cell infusion was offered. OUTCOMES: Although the intracranial metastases did not decrease 10 months after the NK cell treatment, they decreased significantly at 31 months after the treatment and partially disappeared. The tumor response indicated partial response. Furthermore, all of the intracranial metastases continued to decrease at about 42 months after treatment. LESSONS: The brain metastases of NPC are rare with poor prognosis. Radiotherapy in NPC can disrupt the blood-brain barrier, which may contribute to the metastases of brain. This case report will provide rationale for NK cell infusion following regular chemoradiotherapy.

Design, synthesis and structure-activity relationships of novel 15-membered macrolides: Quinolone/quinoline-containing sidechains tethered to the C-6 position of azithromycin acylides.

In the search for novel hybrid molecules by fusing two biologically active scaffolds into one heteromeric chemotype, we found that hybrids of azithromycin and ciprofloxacin/gatifloxacin 26j and 26l can inhibit the supercoiling activity of E. coli gyrase by poisoning it in a way similar to fluoroquinolones. This may modestly contribute to their potencies, which are equal to ciprofloxacin against constitutively resistant Staphylococcus aureus, whose growth is not inhibited by the presence of macrolides. In contrast, introduction of quinolines (the 3-quinoline 26b and the 6-quinoline 26o) with an optimized rigid spacer at the 6-OH of azithromycin acylides did not exert significant potency against constitutively resistant S. aureus, despite the fact that the quinoline-containing compounds, exemplified by 26o, were as active as telithromycin against susceptible, inducibly- and efflux-resistant pathogens. The novel dual modes of action involving protein synthesis inhibition and poisoning DNA replication may pave the way for restoration of antibacterial activities of the current macrolides against constitutively resistant clinical isolates.

Bispecific antibody activated T cells: A newly developed T cells with enhanced proliferation ability and cytotoxicity.

Adoptive cell therapy using ex vivo expanded lymphocytes has shown remarkable efficacy in tumor immunotherapy recently. Among various transfused immune cells, T lymphocytes are the most widely used since they are critical mediators of the immune system and have the capacity to kill tumor cells. However, there are drawbacks in the expanded T cells for transfusion including limited cytotoxicity, limited proliferation and lack of specificity. To improve the quality of these ex vivo expanded T cells, we have designed a new method to expand a group of T cells which are named bispecific antibodies activated T cells. It is the first time that such cells are induced by introducing the bispecific antibody drug (blinatumomab) and feeder cells (normal B cells and irradiated B cell originated lymphoma cells) to the traditional T cells culture system. Culture of freshly isolated human peripheral blood mononuclear cells in this newly designed cell culture system enabled these expanded T cells that (a) displayed a robust proliferation ability; (b) showed fully activated phenotype and enhanced cytokines production; (c) had a low proportion of CD4+CD25+ T regulatory cells and (d) exhibited strengthened cytotoxicity at relatively low effector: target ratios. This work further confirmed the feasibility of rapid induction and expansion of large amounts of human T cells in vitro by using bispecific antibodies and feeder cells. This strategy could also be used for other immune cells rapid expansion and help to improve the quality of these expanded immune cells for adoptive transfusion.

Discovery of hPRDX5-based peptide inhibitors blocking PD-1/PD-L1 interaction through in silico proteolysis and rational design.

PURPOSE: The human peroxiredoxin-5 (hPRDX5) is a member of the family of antioxidant enzymes, which could resist immunosuppression by promoting immune organs development, lymphocyte proliferation and up-regulation of the levels of serum cytokines. However, being a recombinant protein, the hPRDX5 exhibits some problems including the high production cost and bad tissue penetration. Compared to macromolecular therapeutic agents, synthetic peptides have several advantages as drug candidates, such as lower manufacturing costs, reduced immunogenicity, and better organ or tumor penetration. The purpose of this research was to design the novel peptides come from hPRDX5 that can block the interaction of PD-1 and PD-L1. METHODS: Herein in this work, we firstly confirmed the inhibitory activity of hPRDX5 on the binding of PD-L1 to PD-1 based on the previous observation, subsequently, in silico proteolysis and rational design (such as alanine scanning mutagenesis and truncation) were used to automate the design of new peptides derived from hPRDX5 with anti-tumour activity. RESULTS: We found that the most potent peptide could block the PD-1/PD-L1 interaction effectively with an IC50 of 0.646 µM, and could restore the function of Jurkat T cells which had been suppressed by stimulated HCT116 cells. Moreover, experiments with tumor-bearing mice models showed that the peptide IMB-P6-10 could effectively inhibit tumor growth and showed extraordinary low acute toxicity in vivo. CONCLUSIONS: The peptides described in this paper may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

Catalytic Hydrolysis Mechanism of Cocaine by Human Carboxylesterase 1: An Orthoester Intermediate Slows Down the Reaction.

Human carboxylesterase 1 (hCES1) is a major carboxylesterase in the human body and plays important roles in the metabolism of a wide variety of substances, including lipids and drugs, and therefore is attracting more and more attention from areas including lipid metabolism, pharmacokinetics, drug-drug interactions, and prodrug activation. In this work, we studied the catalytic hydrolysis mechanism of hCES1 by the quantum mechanics computation method, using cocaine as a model substrate. Our results support the four-step theory of the esterase catalytic hydrolysis mechanism, in which both the acylation stage and the deacylation stage include two transition states and a tetrahedral intermediate. The roles and cooperation of the catalytic triad, S221, H468, and E354, were also analyzed in this study. Moreover, orthoester intermediates were found in hCES1-catalyzed cocaine hydrolysis reaction, which significantly elevate the free energy barrier and slow down the reaction. Based on this finding, we propose that hCES1 substrates with ß-aminocarboxylester structure might form orthoester intermediates in hCES1-catalyzed hydrolysis, and therefore prolong their in vivo half-life. Thus, this study helps to clarify the catalytic mechanism of hCES1 and elucidates important details of its catalytic process, and furthermore, provides important insights into the metabolism of hCES1 substrates and drug designing.