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The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNA Pyl , has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein. In this work, we show that the C41AU mutant of tRNA Pyl from Candidatus Methanomethylophilus alvus (CmatRNA Pyl ) is catalytically inert toward PylRS from Methanosarcina mazei (MmPylRS) but has weak activity toward PylRS from Ca. M. alvus (CmaPylRS). To improve the catalytic efficiency of CmaPylRS toward CmatRNA Pyl -C41AU, we conducted a directed evolution of CMaPylRS by randomizing its coding sequence, followed by the screening of active mutant clones. After three rounds of randomization and screening, we identified 4 mutations, Y16F/N57D/E161G/N182I, that improve the catalytic efficiency of CMaPylRS toward CMatRNA Pyl -C41AU. This new clone, named R3-14, coupling with CmatRNA Pyl -C41AU to recognize an amber codon, has been successfully used together with an evolved MmPylRS clone, coupling with a mutant M. mazei tRNA Pyl to recognize an ochre codon, to genetically incorporate two different ncAAs, N ε -( t -butoxycarbonyl)-lysine and N ε -acetyl-lysine, into one model protein., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The main protease (M Pro ) of SARS-CoV-2 is crucial for the virus's replication and pathogenicity. Its active site is characterized by four distinct pockets (S1, S2, S4, and S1-3') and a solvent-exposed S3 site for accommodating a protein substrate. During X-ray crystallographic analyses of M Pro bound with dipeptide inhibitors containing a flexible N -terminal group, we often observed an unexpected binding mode. Contrary to the anticipated engagement with the deeper S4 pocket, the N -terminal group frequently assumed a twisted conformation, positioning it for interactions with the S3 site and the inhibitor component bound at the S1 pocket. Capitalizing on this observation, we engineered novel inhibitors to engage both S3 and S4 sites or to adopt a rigid conformation for selective S3 site binding. Several new inhibitors demonstrated high efficacy in M Pro inhibition. Our findings underscore the importance of the S3 site's unique interactions in the design of future M Pro inhibitors as potential COVID-19 therapeutics., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The main protease (M Pro ) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target M Pro 's catalytic cysteine. Our characterization identified potent M Pro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC 50 value of 10 nM against SARS-CoV-2 infection in ACE2 + A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent M Pro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

SARS-CoV-2, the COVID-19 pathogen, relies on its main protease (M Pro ) for replication and pathogenesis. M Pro is a demonstrated target for the development of antivirals for SARS-CoV-2. Past studies have systematically explored tripeptidyl inhibitors such as nirmatrelvir as M Pro inhibitors. However, dipeptidyl inhibitors especially those with a spiro residue at their P2 position have not been systematically investigated. In this work, we synthesized about 30 dipeptidyl M Pro inhibitors and characterized them on enzymatic inhibition potency, structures of their complexes with M Pro , cellular M Pro inhibition potency, antiviral potency, cytotoxicity, and in vitro metabolic stability. Our results indicated that M Pro has a flexible S2 pocket to accommodate inhibitors with a large P2 residue and revealed that dipeptidyl inhibitors with a large P2 spiro residue such as ( S )-2-azaspiro [4,4]nonane-3-carboxylate and ( S )-2-azaspiro[4,5]decane-3-carboxylate have favorable characteristics. One compound, MPI60, containing a P2 ( S )-2-azaspiro[4,4]nonane-3-carboxylate displayed high antiviral potency, low cellular cytotoxicity, and high in vitro metabolic stability.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The novel helper phage allowed for a continuous amber codon enrichment strategy for two different libraries and demonstrated a 100-fold increase in packaging selectivity. CMa13ile40 was then used to create two peptide libraries containing separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine and Nϵ-allyloxycarbonyl-lysine, respectively. These libraries were used to identify peptide ligands that bind to the extracellular domain of ZNRF3. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 that was dependent upon the presence of the ncAA used for selection. Our results demonstrate that ncAAs in phages provide unique interactions for identification of unique peptides. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

As an essential enzyme of SARS-CoV-2, main protease (M Pro ) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M Pro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of M Pro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular M Pro inhibition information to assess an M Pro inhibitor. We used this assay to analyze 30 known M Pro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular M Pro inhibition potency implicating their roles in interfering with key steps other than just the M Pro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular M Pro inhibition IC 50 value of 31 nM that matches closely to its strong antiviral effect with an EC 50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The emergence and rapid spread of SARS-CoV-2, the pathogen of COVID-19, have caused a worldwide public health crisis. The SARS-CoV-2 main protease (Mpro) is an essential enzyme for the virus and therefore an appealing target for the development of antivirals to treat COVID-19 patients. Recently, many in silico screenings have been performed against the main protease to discover novel hits. However, the actual hit rate of virtual screening is often low, and most of the predicted compounds are false positive hits. In this study, we developed a refined virtual screening strategy that incorporated molecular docking and post-docking filtering based on parameters including molecular weight and surface area, aiming to achieve predictions with fewer false positive hits. We applied this strategy to the NCI library containing 284,176 compounds against Mpro. In vitro potency analyses validated several potent inhibitors and thus confirmed the feasibility of our virtual screening strategy. Overall, The study resulted in several potent hit Mpro inhibitors, in which two inhibitors have IC 50 values below 1 μM, that are worth being further optimized and explored. Meanwhile, the refined virtual screen strategy is also applicable to improve general in silico screening hit rates and is useful to accelerate drug discovery for treating COVID-19 and other viral infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Yang, Blankenship, Cho, Xu, Wang and Liu.)

The brazing characteristics and bonding strengths of pure titanium joints are evaluated for joints brazed with Zr-17Ti-22Ni filler. Vacuum brazing was conducted at temperatures between the melting temperatures of the filler metals and the beta-transition temperature of pure titanium at 3 MPa of pressure for 5 min. Fracturing of the pure titanium joint brazed at 1,093 K occurred before yielding during the tensile tests owing to the presence of a serious segregation region containing harder and more brittle [Ti, Zr]2Ni intermetallic compounds. In contrast, in pure titanium joints brazed at and above 1,113 K, fracturing occurred at the base metal. The yield strengths of the samples brazed at 1,113 K-1,133 K were estimated to be in the range of 320-350 MPa and the ultimate tensile strengths likewise ranged from 350 to 380 MPa. The strength of pure titanium brazed at 1,153 K decreased rapidly. The results of this study show that the optimum temperature to ensure good performance after the brazing of pure titanium with Zr-17Ti-22Ni as a filler metal ranges from 1,113 K to 1,133 K.

Purpose: We investigated the gefitinib, 5-fluorouracil (5-FU), leucovorin and oxaliplatin (IFOX) regimen as first-line therapy in patients with metastatic colorectal cancer., Experimental Design: Eligible patients had stage IV colorectal adenocarcinoma, and had not received prior chemotherapy for metastatic disease. Each cycle consisted of 14 days. Cycle 1 consisted of oxaliplatin, leucovorin, and 5-FU (FOLFOX-4). All subsequent cycles consisted of FOLFOX-4 with gefitinib at 500 mg orally daily throughout the 14-day cycle., Results: Forty-five patients were enrolled and were assessable for toxicity. Forty-three patients were assessable for response. Thirty-one of the 43 patients (72%) had either a complete or partial response by the Response Evaluation Criteria in Solid Tumors. Median overall survival was 20.5 months. Median time to progression was 9.3 months. Commonly encountered grade 3 or 4 toxicities included diarrhea in 67% of patients and neutropenia in 60%. Grade 2 acneiform skin rash typical of gefitinib occurred in 60% of patients., Conclusions: IFOX is an active first-line regimen in patients with metastatic colorectal adenocarcinoma, showing higher response rates but also increased toxicities compared with FOLFOX-4 alone in a similar patient population.

The ligament of Treitz suspends the distal duodenum but it has not been identified on abdominal CT scans. Duodenal displacement by an extrinsic mass is not an uncommon finding and is not prevented by the ligament of Treitz. The purpose of this study was to evaluate the size and strength of the ligament of Treitz in autopsy cases and then its visibility on abdominal CT scans. The ligament of Treitz was examined in 18 autopsy cases. The ligament was studied in situ and dissected for macro and micro examination. Size, shape and strength of the ligament were studied. Following the autopsy examination, upper abdominal CT scans were reviewed to identify the ligament. The Ligament of Treitz is a thin membranous and weak structure varying in size and shape. It would not be recognized on a CT image. It would not prevent displacement of the duodenum by an extrinsic mass. Illustrations of the ligament of Treitz in anatomic textbooks often represent an inaccurate picture of the true size and relationship of the ligament to adjacent structures.

Purpose: Aphase 1 study of gefitinib in combination with oxaliplatin, 5-fluorouracil and leucovorin (IFOX)was conducted to evaluate the safety and feasibility of this regimen., Patients and Methods: Patients with advanced solid malignancies were treated with escalating doses of gefitinib (250 mg or 500 mg once daily) in combination with FOLFOX (oxaliplatin, 5-fluorouracil, and leucovorin). The initial dose of oxaliplatin was 70 mg/m2 with sequential dose escalation to 85 mg/m2., Results: Sixteen patients received a total of 138 14-day courses of daily gefitinib in combination with FOLFOX. Escalation of gefitinib from 250 mg/d to 500 mg/d with FOLFOX was well-tolerated. In addition, no severe toxicities precluded subsequent dose escalation of oxaliplatin from 70 mg/m2 to 85 mg/m2 at which no dose-limiting toxicity was seen. No further dose escalation was performed as this represented the oxaliplatin dose administered in the standard FOLFOX-4 regimen. The most predominant toxicity was diarrhea, which was well controlled with oral antidiarrheal agents. Four partial remissions occurred in patients with metastatic colorectal cancer., Conclusions: Gefitinib as a 500 mg daily continuous dose was well tolerated in combination with full doses of FOLFOX-4.

Purpose: To investigate the gefitinib, fluorouracil (FU), leucovorin, and oxaliplatin regimen (IFOX) in previously treated patients with metastatic colorectal cancer., Patients and Methods: Eligible patients had stage IV colorectal adenocarcinoma and had demonstrated progression or intolerance to a prior chemotherapy regimen not including oxaliplatin. Each cycle consisted of 14 days. Cycle 1 consisted of oxaliplatin 85 mg/m2 intravenously (IV) during 2 hours on day 1, hours 0 to 2; leucovorin 200 mg/m2 IV on days 1 and 2, hours 0 to 2; FU 400 mg/m2 IV push on days 1 and 2; and FU 600 mg/m2 IV on days 1 and 2, hours 2 to 24 (FOLFOX-4). All subsequent cycles consisted of FOLFOX-4 with gefitinib at 500 mg/d administered orally throughout the 14-day cycle., Results: Twenty-seven patients were enrolled onto the study. The median number of prior chemotherapy regimens was two, and 74% of all patients received prior irinotecan. Nine of the 27 patients (33%) and six of the 20 patients (30%) who had prior FU and irinotecan had a partial response by Response Evaluation Criteria in Solid Tumors Group criteria. Median overall survival was 12.0 months. Median event-free survival was 5.4 months. Grade 3 to 4 toxicities included neutropenia (48%), diarrhea (48%), nausea (22%), and vomiting (15%)., Conclusion: IFOX is an active regimen in patients with previously treated metastatic colorectal adenocarcinoma, demonstrating higher response rates than those reported with FOLFOX-4 alone in a similar patient population.

Purpose: The purpose of this study was to establish the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), and preliminary activity of BMS-188797 administered weekly., Experimental Design: Patients with advanced malignancies were treated with escalating doses of BMS-188797 on a weekly schedule as a 1-h i.v. infusion. Plasma sampling was performed to characterize the pharmacokinetics of BMS-188797., Results: Eighteen patients with advanced malignancies were enrolled at three dose levels ranging from 35 to 65 mg/m(2). The number of patients evaluated at each dose level was as follows: 35 mg/m(2) (n = 3); 50 mg/m(2) (n = 9); and 65 mg/m(2) (n = 6). At 65 mg/m(2), three of six patients had a DLT (one had grade 4 neutropenia lasting >7 days, and two had grade 3 diarrhea). Expansion of the 50-mg/m(2) dose cohort to nine patients established this dose as the MTD, with one patient experiencing a DLT (grade 4 neutropenia with fever). Two partial responses were observed (lung cancer, 7+ months; ovarian cancer, 6+ months durations), as well as two minor responses (esophageal cancer, 5 months; ovarian cancer, 5 months). Both patients with partial responses had been clinically resistant to paclitaxel. Plasma pharmacokinetic mean values of maximum concentration (C(max)) and area under the curve (AUC(0-48)) increased in a dose-dependent manner within the range of doses used in this study, and in three of four patients, the DLTs correlated with AUC., Conclusions: The MTD and the recommended Phase II dose of weekly BMS-188797 is 50 mg/m(2). The drug demonstrates antitumor activity in taxane-refractory solid tumors and is now being evaluated in combination with carboplatin.

Blood and urine samples were collected simultaneously with measurements of pulmonary function at 2-hour intervals for 8 hours after oral administration of short-acting (SAT) and long-acting theophylline (LAT) preparations in 15 patients with stable chronic obstructive lung disease (COLD) on long-term maintenance theophylline therapy. The relationship between pulmonary function tests, serum theophylline level, plasma and urinary adenosine 3'5' cyclic monophosphate (cAMP) was examined. The highest forced expiratory volume in one second FEV1 was obtained with STL of 12.8 micrograms/ml +/- 5.21 SD and 9.14 micrograms/ml +/- 6.15 (P less than .05) after administration of SAT and LAT, respectively. A further increase in serum theophylline level (STL) offered no therapeutic benefit, and, in fact, was associated with a fall in FEV1 in many instances. Plasma or urinary cAMP measurements did not correlate with STL. STL at which the best pulmonary function is achieved is quite variable in patients with COLD on maintenance theophylline therapy, is frequently less than 10 micrograms/ml, and can be determined only by repeated measurements of pulmonary function at different STL. Therefore measurements of STL are of limited value in guiding treatment with theophylline.

The protective effect of propylthiouracil (PTU) pretreatment against acetaminophen-induced erythrocyte osmotic fragility was determined in the male Fisher rat. Hepatotoxicity was assessed for comparative purposes. PTU (0.15%) was fed in chow for a period of 12 days. Acetaminophen (1 g/kg body wt) was then administered orally by a stomach tube after an overnight fast. The rats were killed either 4 or 24 hr later. Erythrocyte osmotic fragility was determined by the extent of hemolysis in various concentrations of NaCl solutions. Hepatotoxicity was assessed by a rise in serum transaminases and by histological examination of hepatic tissue. PTU treatment when compared with control not only protected rats against acetaminophen-induced hepatotoxicity as reported before, but also protected against erythrocyte osmotic fragility. The time course of acetaminophen toxicity seems to be similar for liver and erythrocyte since both showed damage after 24 hr but not after 4 hr of acetaminophen administration. The data show that PTU pretreatment affords protection against acetaminophen-induced increased erythrocyte osmotic fragility even when their glutathione concentrations were not significantly different, suggesting that PTU per se has a protective effect.

The effect of pair feeding of euthyroid rats compared with propylthiouracil (PTU) treated rats on acetaminophen (APAP)-induced hepatotoxicity was studied. Also, the effect of food deprivation of both the euthyroid and PTU-induced hypothyroid rats for 24 h, as well as forced feeding of the euthyroid rats after a toxic dose of APAP, was determined. Pair feeding decreased both protein and energy intake compared with ad libitum-fed controls and resulted in decreased growth rate similar to that for the PTU treated rats. In contrast to the protective effect of PTU pretreatment, decreased protein energy intake by the euthyroid rats either tended to make them more susceptible to acetaminophen-induced hepatotoxicity or had no effect as assessed by elevation of serum transaminases (SGOT,SGPT) and by hepatic necrotic score. Pair feeding also significantly altered drug disposition with an increase in the molar ratio of urinary APAP-mercapturic acid conjugate, but not the absolute amount, suggesting possible increased cytochrome P-450 dependent drug metabolizing enzyme activity. Compared with PTU-fed, in the pair-fed the molar ratio of glucuronide conjugate decreased and sulfate conjugate increased. Hepatic reduced glutathione (GSH) concentrations before and 4 h after a toxic dose of acetaminophen administration were higher in the PTU pretreated compared with euthyroid rats. Fasting of the PTU pretreated rats for 24 h after acetaminophen administration abolished the PTU-induced protective effect. Forced feeding of the euthyroid rats after a toxic dose of acetaminophen increased rather than decreased the toxicity when compared with euthyroid ad libitum-fed rats. Data suggest that higher concentrations of hepatic glutathione in the PTU pretreated compared with euthyroid rats before and 4 h after acetaminophen administration contribute to PTU-induced protection. Forced feeding of rats when the liver is severely damaged and its function compromised is harmful rather than protective. We conclude that the nutritional state of the animal significantly influences drug toxicity and should be taken into consideration in designing drug therapy and evaluation of drug toxicity.