ABSTRACT
The COVID-19 pandemic has uncovered the high genetic variability of the SARS-CoV-2 virus and its ability to evade the immune responses that were induced by earlier viral variants. Only a few monoclonal antibodies that have been reported to date are capable of neutralizing a broad spectrum of SARS-CoV-2 variants. Here, we report the isolation of a new broadly neutralizing human monoclonal antibody, iC1. The antibody was identified through sorting the SARS-CoV-1 RBD-stained individual B cells that were isolated from the blood of a vaccinated donor following a breakthrough infection. In vitro, iC1 potently neutralizes pseudoviruses expressing a wide range of SARS-CoV-2 Spike variants, including those of the XBB sublineage. In an hACE2-transgenic mouse model, iC1 provided effective protection against the Wuhan strain of the virus as well as the BA.5 and XBB.1.5 variants. Therefore, iC1 can be considered as a potential component of the broadly neutralizing antibody cocktails resisting the SARS-CoV-2 mutation escape.
Subject(s)
Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Mice, Transgenic , SARS-CoV-2 , Animals , SARS-CoV-2/immunology , Humans , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Mice , Antibodies, Viral/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Pandemics/prevention & control , Betacoronavirus/immunology , Betacoronavirus/genetics , Broadly Neutralizing Antibodies/immunology , Disease Models, Animal , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Pneumonia, Viral/prevention & control , Coronavirus Infections/immunology , Coronavirus Infections/virology , Coronavirus Infections/prevention & controlABSTRACT
The major protective immune response against viruses is the production of type I and III interferons (IFNs). IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs) that block viral replication and further viral spread. In this report, we analyzed the expression of IFNs and some ISGs (MxA, PKR, OAS-1, IFIT-1, RIG-1, MDA5, SOCS-1) in alveolar epithelial cells (A549) in response to infection with influenza A viruses (A/California/07/09 (H1N1pdm); A/Texas/50/12 (H3N2)); influenza B virus (B/Phuket/3073/13); adenovirus type 5 and 6; or respiratory syncytial virus (strain A2). Influenza B virus had the ability to most rapidly induce IFNs and ISGs as well as to stimulate excessive IFN-α, IFN-ß and IFN-λ secretion. It seems curious that IAV H1N1pdm did not induce IFN-λ secretion, but enhanced type I IFN and interleukin (IL)-6 production. We emphasized the importance of the negative regulation of virus-triggered signaling and cellular IFN response. We showed a decrease in IFNLR1 mRNA in the case of IBV infection. The attenuation of SOCS-1 expression in IAV H1N1pdm can be considered as the inability of the system to restore the immune status. Presumably, the lack of negative feedback loop regulation of proinflammatory immune response may be a factor contributing to the particular pathogenicity of several strains of influenza. Keywords: lambda interferons; MxA; influenza; respiratory syncytial virus; A549 cells.
Subject(s)
Influenza, Human , Interferon Lambda , Humans , Influenza, Human/genetics , Influenza A Virus, H3N2 Subtype , Interferons/genetics , Interferons/pharmacology , Interferon-alpha/genetics , Gene ExpressionABSTRACT
Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Humans , Vaccines, Inactivated , COVID-19/prevention & control , COVID-19 Serotherapy , COVID-19 Vaccines , Pandemics , Propiolactone/pharmacology , SARS-CoV-2 , FormaldehydeABSTRACT
Immune evasion of SARS-CoV-2 undermines current strategies tocounteract the pandemic, with the efficacy of therapeutic virus-neutralizing monoclonal antibodies (nAbs) being affected the most. In this work, we asked whether two previously identified human cross-neutralizing nAbs, iB14 (class VH1-58) and iB20 (class VH3-53/66), are capable of neutralizing the recently emerged Omicron (BA.1) variant. Both nAbs were found to bind the Omicron RBD with a nanomolar affinity, yet they displayed contrasting functional features. When tested against Omicron, the neutralizing activity of iB14 was reduced 50-fold, whereas iB20 displayed a surprising increase in activity. Thus, iB20 is a unique representative of the VH3-53/66-class of nAbs in terms of breadth of neutralization, which establishes it as a candidate for COVID-19 therapy and prophylactics.
ABSTRACT
Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , Disulfides , Epitopes , Humans , Immunization , Mice , Recombinant Proteins/genetics , SARS-CoV-2ABSTRACT
In this study, we assessed in humans the immunogenicity and safety of one dose (7.5 or 15 µg of hemagglutinin [HA]) of a whole-virion inactivated prepandemic influenza vaccine adjuvanted with aluminum hydroxide. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/Chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and microneutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients, with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 after a dose of 7.5 µg in the HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 µg was used. The percentages of individuals achieving anti-HA titers of ≥1:40 were 52.5% and 57.5% for the 7.5- and 15-µg dose groups, respectively. Similar results were obtained when antibodies were analyzed in an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in the two dose groups, respectively) were detected against heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole-virion prepandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/adverse effects , Influenza A Virus, H5N1 Subtype/immunology , Influenza Vaccines/adverse effects , Influenza Vaccines/immunology , Influenza, Human/prevention & control , Adult , Aluminum Hydroxide/administration & dosage , Aluminum Hydroxide/adverse effects , Animals , Antibodies, Viral/blood , Cross Reactions , Hemagglutination Inhibition Tests , Humans , Influenza Vaccines/administration & dosage , Influenza, Human/virology , Kazakhstan , Male , Middle Aged , Neutralization Tests , Reverse Genetics , Russia , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/adverse effects , Vaccines, Inactivated/immunology , Young AdultABSTRACT
Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.
Subject(s)
Mass Spectrometry , Protein Phosphatase 1/metabolism , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Catalytic Domain , DNA-Binding Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/metabolism , Mutant Proteins/metabolism , Nuclear Proteins/metabolism , Phosphorylation , Protein Structure, Tertiary , RNA-Binding Proteins/metabolismABSTRACT
The discovery of 5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC50 <0.10 microM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.
Subject(s)
Antiviral Agents/chemistry , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Pyridones/chemistry , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Macaca fascicularis , Microsomes, Liver/metabolism , Pyridones/chemical synthesis , Pyridones/pharmacology , RNA-Dependent RNA Polymerase/metabolism , Structure-Activity RelationshipABSTRACT
A novel series of non-nucleoside small molecules containing a tricyclic dihydropyridinone structural motif was identified as potent HCV NS5B polymerase inhibitors. Driven by structure-based design and building on our previous efforts in related series of molecules, we undertook extensive SAR studies, in which we identified a number of metabolically stable and very potent compounds in genotype 1a and 1b replicon assays. This work culminated in the discovery of several inhibitors, which combined potent in vitro antiviral activity against both 1a and 1b genotypes, metabolic stability, good oral bioavailability, and high C(12) (PO)/EC(50) ratios.
Subject(s)
Biological Availability , Drug Design , Structure-Activity Relationship , Antiviral Agents/pharmacokinetics , Chemistry, Pharmaceutical , Crystallography, X-Ray , Drug Evaluation, Preclinical , Genotype , Hepacivirus/drug effects , Hepatitis C , Molecular Structure , RNA-Dependent RNA Polymerase , Viral Nonstructural Proteins/antagonists & inhibitorsABSTRACT
5,6-Dihydro-1H-pyridin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4ad displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; IC(50) (1a)<25nM, EC(50) (1b)=16nM), good in vitro DMPK properties, as well as moderate oral bioavailability in monkeys (F=24%).
Subject(s)
DNA-Directed RNA Polymerases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Pyridones/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Haplorhini , Pyridones/administration & dosage , Pyridones/chemistry , Pyridones/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.
Subject(s)
Antiviral Agents/chemical synthesis , Chemistry, Pharmaceutical/methods , Cyclic S-Oxides/chemical synthesis , Pyridazines/chemistry , Pyridazines/chemical synthesis , Thiadiazines/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Antiviral Agents/pharmacology , Caco-2 Cells , Crystallography, X-Ray/methods , Cyclic S-Oxides/pharmacology , DNA-Directed RNA Polymerases/chemistry , Drug Design , Genotype , Humans , Inhibitory Concentration 50 , Microsomes/metabolism , Models, Chemical , Molecular Conformation , Pyridazines/pharmacology , Structure-Activity Relationship , Thiadiazines/pharmacologyABSTRACT
Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b) <10 nM; EC(50) (1b)=34 nM) as well as good stability towards human liver microsomes (HLM t(1/2) =59 min).
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Hepacivirus/drug effects , Microsomes, Liver/drug effects , Pyridazines/chemical synthesis , Pyridazines/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Drug Design , Humans , Molecular Structure , Pyridazines/chemistry , Structure-Activity RelationshipABSTRACT
4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).
Subject(s)
Chemistry, Pharmaceutical/methods , Hepacivirus/enzymology , Microsomes, Liver/enzymology , Pyridazines/chemical synthesis , Pyridazines/pharmacology , Thiazines/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Caco-2 Cells , Crystallography, X-Ray/methods , Drug Design , Hepacivirus/drug effects , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , Pyridazines/chemistry , Structure-Activity Relationship , Thiazines/chemistry , Thiazines/pharmacology , Time FactorsABSTRACT
A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC(50) values. The most potent compound exhibited IC(50) less than 10nM against the genotype 1b HCV polymerase and EC(50) of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacokinetics , Enzyme Inhibitors/pharmacokinetics , Thiazoles/chemical synthesis , Thiophenes/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Crystallography, X-Ray/methods , Drug Design , Genotype , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , RNA, Viral/metabolism , Structure-Activity Relationship , Thiazoles/pharmacokinetics , Thiophenes/pharmacokineticsABSTRACT
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacokinetics , Hepacivirus/drug effects , Pyridazines/chemical synthesis , Pyridazines/pharmacokinetics , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Animals , Antiviral Agents/blood , Antiviral Agents/chemistry , Combinatorial Chemistry Techniques , Drug Design , Humans , Microsomes, Liver/drug effects , Molecular Structure , Pyridazines/blood , Pyridazines/chemistry , Rats , Structure-Activity RelationshipABSTRACT
Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).
Subject(s)
Antiviral Agents/pharmacology , Pyridazines/pharmacology , Pyrroles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Binding Sites/drug effects , Cell Line , Crystallography, X-Ray , Humans , Hydrogen Bonding , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Pyridazines/chemical synthesis , Pyridazines/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistryABSTRACT
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as potent inhibitors of genotype 1 HCV NS5B polymerase focusing on the optimization of their drug metabolism and pharmacokinetics (DMPK) profiles. This investigation led to the discovery of potent inhibitors with improved DMPK properties.
Subject(s)
Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacokinetics , Hepacivirus/enzymology , Pyridazines/chemical synthesis , Pyridazines/pharmacokinetics , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Animals , Antiviral Agents/chemistry , Combinatorial Chemistry Techniques , Drug Design , Haplorhini , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Pyridazines/chemistry , Structure-Activity RelationshipABSTRACT
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The synthesis, structure-activity relationships (SAR), metabolic stability, and structure-based design approach for this new class of compounds are discussed.
Subject(s)
Benzothiadiazines/chemistry , Benzothiadiazines/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyridazines/chemistry , Pyridazines/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Benzothiadiazines/metabolism , Crystallography, X-Ray , Drug Design , Drug Stability , Enzyme Inhibitors/metabolism , Humans , Hydrogen Bonding , Inhibitory Concentration 50 , Microsomes, Liver/metabolism , Models, Molecular , Pyridazines/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The structure-activity relationship (SAR) associated with variation of the pyridazinone 2- and 6-substituents is discussed. The synthesis and metabolic stability of this new class of compounds are also described.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyridazines/chemistry , Pyridazines/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Drug Stability , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Inhibitory Concentration 50 , Microsomes, Liver/metabolism , Models, Molecular , Pyridazines/chemical synthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Thiadiazines/chemical synthesis , Thiadiazines/chemistry , Thiadiazines/pharmacology , Viral Nonstructural Proteins/metabolismABSTRACT
We have determined the kinetic parameters of human recombinant thymidylate synthase (hrTS) with its natural substrate, dUMP, and E-5-(2-bromovinyl)-2(')-deoxyuridine monophosphate (BVdUMP), a nucleotide derivative believed to be the active species of the novel anticancer drug NB1011. NB1011 is activated by hrTS and is selectively toxic to high thymidylate synthase expressing tumor cells. BVdUMP undergoes hrTS-catalyzed thiol-dependent transformation. dUMP and BVdUMP act as competitive hrTS substrates. The natural folate cofactor, CH(2)-THF, inhibits the TS-catalyzed reaction with BVdUMP. We suggest that lower folate levels found in tumor cells favor TS-catalyzed BVdUMP transformation, which, in addition to higher levels of TS expression in tumor cells, contributes to the favorable therapeutic index of the drug NB1011.