Distinguishing between the two enantiomers of a molecule is a challenging task due to their nearly identical physical properties. Time-consuming chromatography methods are typically required for this ...task, which greatly limits the throughput of analysis. Here we describe a fluorescence-based method for the rapid and high-throughput analysis of both small-molecule enantiopurity and concentration. Our approach relies on selective molecular recognition of one enantiomer of the target molecule using a DNA aptamer, and the ability of aptamer-based biosensors to transduce the presence of a target molecule into a dose-dependent fluorescence signal. The key novel aspect of our approach is the implementation of enantiomeric DNA biosensors, which are synthesized from d- and l-DNA, but labeled with orthogonal fluorophores. According to the principle of reciprocal chiral substrate specificity, these biosensors will bind to opposite enantiomers of the target with equal affinity and selectivity, enabling simultaneous quantification of both enantiomers of the target. Using the previously reported DNA biosensor for l-tyrosinamide (l-Tym), we demonstrate the ability to rapidly and accurately measure both enantiopurity and concentration for mixtures of l- and d-Tym. We also apply our enantiomeric biosensors to the optimization of reaction conditions for the synthesis of d-Tym and provide mathematical modeling to suggest that DNA biosensors having only modest binding selectivity can also be used for fluorescence-based enantiopurity measurement. This research provides a generalizable method for high-throughput analysis of reaction mixtures, which is anticipated to significantly accelerate reaction optimization for the synthesis of high-value chiral small molecules.
Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved ...therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without ...biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.
The methylotrophic yeast Pichia pastoris (reclassified as Komagataella phaffii) is a versatile protein expression system, yet many commonly used promoters have attributes undesirable for fermentation ...or its optimization. Hence, the copper‐inducible CUP1 gene promoter from the related yeast Saccharomyces cerevisiae was used to express human gelatin. Multimerization of a potential copper response element in the CUP1 promoter, a S. cerevisiae Ace1p binding site, significantly increased gelatin expression. Expression was induced by copper in a dose‐dependent fashion and was not dependent on cell density. Gelatin was additionally induced in standard copper‐containing fermentation basal salts media. Removal of a S. cerevisiae heat shock factor (Hsf1p) binding site reduced copper‐dependent gelatin induction suggesting that a similar protein may regulate this promoter in P. pastoris. This engineered copper inducible promoter expands the yeast recombinant protein production tool kit.
Take Away
Multimerization of the Ace1p binding site in the of S. cerevisiae CUP1 promoter in a human gelatin expression vector significantly increased copper‐dependent expression in P. pastoris.
This engineered promoter has desirable attributes for fermentation as copper dose‐dependently induced gelatin expression at a range of cell densities in standard fermentation media.
The P. pastoris homolog of S. cerevisiae heat shock factor (Hsf1p) may regulate this promoter as removal of the Hsf1p binding site reduced the induction of gelatin in a copper‐dependent fashion in P. pastoris.
BACKGROUND:Intrathecal opioids are routinely administered during spinal anesthesia for postcesarean analgesia. The effectiveness of intrathecal morphine for postcesarean analgesia is well ...established, and the use of intrathecal hydromorphone is growing. No prospective studies have compared the effectiveness of equipotent doses of intrathecal morphine versus intrathecal hydromorphone as part of a multimodal analgesic regimen for postcesarean analgesia. The authors hypothesized that intrathecal morphine would result in superior analgesia compared with intrathecal hydromorphone 24 h after delivery.
METHODS:In this single-center, double-blinded, randomized trial, 138 parturients undergoing scheduled cesarean delivery were randomized to receive 150 µg of intrathecal morphine or 75 µg of intrathecal hydromorphone as part of a primary spinal anesthetic and multimodal analgesic regimen; 134 parturients were included in the analysis. The primary outcome was the numerical rating scale score for pain with movement 24 h after delivery. Static and dynamic pain scores, nausea, pruritus, degree of sedation, and patient satisfaction were assessed every 6 h for 36 h postpartum. Total opioid consumption was recorded.
RESULTS:There was no significant difference in pain scores with movement at 24 h (intrathecal hydromorphone median 25th, 75th 4 3, 5 and intrathecal morphine 3 2, 4.5) or at any time point (estimated difference, 0.5; 95% CI, 0 to 1; P = 0.139). Opioid received in the first 24 h did not differ between groups (median 25th, 75th oral morphine milligram equivalents for intrathecal hydromorphone 30 7.5, 45.06 vs. intrathecal morphine 22.5 14.0, 37.5, P = 0.769). From Kaplan–Meier analysis, the median time to first opioid request was 5.4 h for hydromorphone and 12.1 h for morphine (log-rank test P = 0.200).
CONCLUSIONS:Although the hypothesis was that intrathecal morphine would provide superior analgesia to intrathecal hydromorphone, the results did not confirm this. At the doses studied, both intrathecal morphine and intrathecal hydromorphone provide effective postcesarean analgesia when combined with a multimodal analgesia regimen.
The
drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate
drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used ...for the treatment of a range of bacterial infections, is not active against
. Thus, evidence that
DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against
, exerts whole cell activity against
. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of
DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in
. As observed in
, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against
. PQD-1 is active against subspecies of
and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify
DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in
.
The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency ...profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK‐4882 2‐((S)‐pyrrolidin‐2‐yl)‐5‐(2‐(4‐(5‐((S)‐pyrrolidin‐2‐yl)‐1H‐imidazol‐2‐yl)phenyl)benzofuran‐5‐yl)‐1H‐imidazole. While preclinical proof‐of‐concept studies in HCV‐infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single‐ and multiple‐dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK‐4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK‐8742, a tetracyclic indole‐based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all‐oral, interferon‐free regimen for the treatment of HCV infection.
Effective treatment of chronic hepatitis C with direct‐acting antivirals will require combination therapy with multiple agents that target different steps in the viral replication cycle and impose a high barrier to resistance. MK‐8742 is a potent inhibitor of hepatitis C virus non‐structural protein 5A (HCV NS5A) that is being developed as a component of an once‐daily, all‐oral, interferon‐free regimen for the treatment of chronic HCV infection.
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