ABSTRACT
BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP) inhibitors (PARPi) exploit tumour-specific defects in homologous recombination DNA repair and continuous dosing is most efficacious. Early clinical trial data with rucaparib suggested that it caused sustained PARP inhibition. Here we investigate the mechanism of this durable inhibition and potential exploitation. METHODS: Uptake and retention of rucaparib and persistence of PARP inhibition were determined by radiochemical and immunological assays in human cancer cell lines. The pharmacokinetics and pharmacodynamics of rucaparib were determined in tumour-bearing mice and the efficacy of different schedules of rucaparib was determined in mice bearing homologous recombination DNA repair-defective tumours. RESULTS: Rucaparib accumulation is carrier mediated (Km=8.4±1.2 µM, Vmax=469±22 pmol per 10(6) cells per 10 min), reaching steady-state levels >10 times higher than the extracellular concentration within 30 min. Rucaparib is retained in cells and inhibits PARP ≥50% for ≥72 h days after a 30-min pulse of 400 nM. In Capan-1 tumour-bearing mice rucaparib accumulated and was retained in the tumours, and PARP was inhibited for 7 days following a single dose of 10 mg kg(-1) i.p or 150 mg kg(-1) p.o. by 70% and 90%, respectively. Weekly dosing of 150 mg kg(-1) p.o once a week was as effective as 10 mg kg(-1) i.p daily for five days every week for 6 weeks in delaying Capan-1 tumour growth. CONCLUSIONS: Rucaparib accumulates and is retained in tumour cells and inhibits PARP for long periods such that weekly schedules have equivalent anticancer activity to daily dosing in a pre-clinical model, suggesting that clinical evaluation of alternative schedules of rucaparib should be considered.
Subject(s)
Enzyme Inhibitors/administration & dosage , Indoles/administration & dosage , Poly(ADP-ribose) Polymerases/genetics , Animals , Cell Line, Tumor , DNA Repair/drug effects , Drug Administration Schedule , Enzyme Inhibitors/blood , Enzyme Inhibitors/pharmacokinetics , Homologous Recombination/drug effects , Humans , Indoles/blood , Indoles/pharmacokinetics , Mice , Poly(ADP-ribose) Polymerase Inhibitors , Xenograft Model Antitumor AssaysABSTRACT
The stress-inducible transcription factor, nuclear factor (NF)-κB induces genes involved in proliferation and apoptosis. Aberrant NF-κB activity is common in cancer and contributes to therapeutic-resistance. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated during DNA strand break repair and is a known transcriptional co-regulator. Here, we investigated the role of PARP-1 function during NF-κB activation using p65 small interfering RNA (siRNA), PARP siRNA or the potent PARP-1 inhibitor, AG-014699. Survival and apoptosis assays showed that NF-κB p65(-/-) cells were more sensitive to ionizing radiation (IR) than p65(+/+) cells. Co-incubation with p65 siRNA, PARP siRNA or AG-014699 radio-sensitized p65(+/+), but not p65(-/-) cells, demonstrating that PARP-1 mediates its effects on survival via NF-κB. Single-strand break (SSB) repair kinetics, and the effect SSB repair inhibition by AG-014699 were similar in p65(+/+) and p65(-/-) cells. As preventing SSB repair did not radio-sensitize p65(-/-) cells, we conclude that radio-sensitization by AG-014699 is due to downstream inhibition of NF-κB activation, and independent of SSB repair inhibition. PARP-1 catalytic activity was essential for IR-induced p65 DNA binding and NF-κB-dependent gene transcription, whereas for tumor necrosis factor (TNF)-α-treated cells, PARP-1 protein alone was sufficient. We hypothesize that this stimulus-dependent differential is mediated via stimulation of the poly(ADP-ribose) polymer, which was induced following IR, not TNF-α. Targeting DNA damage-activated NF-κB using AG-014699 may therefore overcome toxicity observed with classical NF-κB inhibitors without compromising other vital inflammatory functions. These data highlight the potential of PARP-1 inhibitors to overcome NF-κB-mediated therapeutic resistance and widens the spectrum of cancers in which these agents may be utilized.
Subject(s)
Enzyme Inhibitors/pharmacology , Indoles/pharmacology , NF-kappa B/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Radiation Tolerance , Animals , Cell Line , Infrared Rays , Mice , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , RNA, Small Interfering , Tumor Necrosis Factor-alpha/administration & dosageABSTRACT
BACKGROUND: Temozolomide shows activity against medulloblastoma, the most common malignant paediatric brain tumour. Poly(ADP-ribose) polymerase (PARP) inhibitors enhance temozolomide activity in extracranial adult and paediatric human malignancies. METHODS: We assessed the effect of AG-014699, a clinically active PARP inhibitor, on temozolomide-induced growth inhibition in human medulloblastoma models. Pharmacokinetic, pharmacodynamic and toxicity assays were performed in tumour-bearing mice. RESULTS: Sensitivity to temozolomide in vitro was consistent with methylguanine methyltransferase (MGMT) and DNA mismatch repair (MMR) status; MGMT(+) MMR(+) D384Med cells (temozolomide GI(50)=220 µM), representative of most primary medulloblastomas, were sensitised fourfold by AG-014699; MGMTâ» MMR(+) D425Med cells were hypersensitive (GI(50)=9 µM) and not sensitised by AG-014699, whereas MGMT(+) MMRâ» temozolomide-resistant D283Med cells (GI50=807 µM) were sensitised 20-fold. In xenograft models, co-administration of AG-014699 produced an increase in temozolomide-induced tumour growth delay in D384Med xenografts. Consistent with the in vitro data, temozolomide caused complete tumour regressions of D425Med xenografts, whereas D283Med xenografts were relatively resistant. AG-014699 was not toxic, accumulated and reduced PARP activity ≥75% in xenograft and brain tissues. CONCLUSION: We show for the first time central nervous system penetration and inhibition of brain PARP activity by AG-014699. Taken together with our in vitro chemosensitisation and toxicity data, these findings support further evaluation of the clinical potential of AG-014699-temozolomide combinations in intra-cranial malignancies.
Subject(s)
Antineoplastic Agents, Alkylating/therapeutic use , Central Nervous System Neoplasms/pathology , Dacarbazine/analogs & derivatives , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Cell Division/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Central Nervous System Neoplasms/drug therapy , Central Nervous System Neoplasms/enzymology , Child , DNA Mismatch Repair/drug effects , DNA Repair/drug effects , Dacarbazine/therapeutic use , Humans , Indoles/therapeutic use , Medulloblastoma/drug therapy , Medulloblastoma/enzymology , Medulloblastoma/pathology , Mice , Mice, Nude , Poly (ADP-Ribose) Polymerase-1 , Protein Serine-Threonine Kinases/antagonists & inhibitors , Temozolomide , Transplantation, HeterologousABSTRACT
The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, K(i) = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit K(i) values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, K(i) = 1.6 nM) being one of the most potent. Insight into structure-activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, K(i) = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, K(i) = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).
Subject(s)
Benzimidazoles/chemistry , Benzimidazoles/chemical synthesis , Dacarbazine/analogs & derivatives , Enzyme Inhibitors/chemical synthesis , Poly(ADP-ribose) Polymerase Inhibitors , Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Crystallography, X-Ray , Dacarbazine/pharmacology , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Drug Synergism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Structure-Activity Relationship , Temozolomide , Topotecan/pharmacology , Tumor Cells, CulturedABSTRACT
Potent poly(ADP-ribose) polymerase (PARP) inhibitors have been developed that potentiate the cytotoxicity of ionizing radiation and anticancer drugs. The biological effects of two novel PARP inhibitors, NU1025 (8-hydroxy-2-methylquinazolin-4-[3H]one, Ki = 48 nM) and NU1085 [2-(4-hydroxyphenyl)benzamidazole-4-carboxamide, Ki = 6 nM], in combination with temozolomide (TM) or topotecan (TP) have been studied in 12 human tumor cell lines (lung, colon, ovary, and breast cancer). Cells were treated with increasing concentrations of TM or TP +/- NU1025 (50, 200 microM) or NU1085 (10 microM) for 72 h. The potentiation of growth inhibition by NU1025 and NU1085 varied between the cell lines from 1.5- to 4-fold for TM and 1- to 5-fold for TP and was unaffected by p53 status. Clonogenic assays undertaken in two of the cell lines confirmed that the potentiation of growth inhibition reflected the potentiation of cytotoxicity. NU1025 (50 microM) was about as effective as 10 microM NU1085 at potentiating growth inhibition and cytotoxicity, consistent with the relative potencies of the two molecules as PARP inhibitors. Potentiation of cytotoxicity was obtained at concentrations of NU1025 and NU1085 that were not toxic per se; however, NU1085 alone was 3-fold more cytotoxic (LC50 values ranged from 83 to 94 microM) than NU1025 alone (LC50 > 900 microM). These data demonstrate that PARP inhibitors are effective resistance-modifying agents in human tumor cell lines and have provided a comprehensive assessment protocol for the selection of optimum combinations of anticancer drugs, PARP inhibitors, and cell lines for in vivo studies.
Subject(s)
Antineoplastic Agents/toxicity , Cell Division/drug effects , Cell Survival/drug effects , Dacarbazine/analogs & derivatives , Enzyme Inhibitors/toxicity , Poly(ADP-ribose) Polymerase Inhibitors , Quinazolines/toxicity , Topotecan/toxicity , Breast Neoplasms , Colonic Neoplasms , Dacarbazine/toxicity , Drug Synergism , Female , Humans , Lung Neoplasms , Ovarian Neoplasms , Temozolomide , Tumor Cells, Cultured , Tumor Stem Cell AssayABSTRACT
In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.
Subject(s)
DNA Repair , Poly(ADP-ribose) Polymerases/metabolism , 3T3 Cells , Animals , Base Sequence , Cells, Cultured , DNA Polymerase beta/metabolism , DNA Primers , Mice , NAD/metabolism , Poly(ADP-ribose) Polymerases/geneticsABSTRACT
BACKGROUND: Hepatitis C virus (HCV) NS3 proteinase activity is required for the release of HCV nonstructural proteins and is thus a potential antiviral target. The enzyme requires a protein cofactor NS4A, located downstream of NS3 on the polyprotein, for activation and efficient processing. OBJECTIVES: Comparison of the proteinase three-dimensional structure before and after NS4A binding should help to elucidate the mechanism of NS4A-dependent enzyme activation. STUDY DESIGN: We determined the crystal structure of NS3 proteinase of HCV BK isolate (genotype 1b; residues 1-189) and also the crystal structure of this proteinase complexed with HCV BK-NS4A (residues 21-34). RESULTS: The core region (residues 30-178) of the enzyme without cofactor (NS3P) or with bound cofactor (NS3P/4A) is folded into a trypsin-like conformation and the substrate P1 specificity pocket is essentially unchanged. However, the D1-E1 beta-loop shifts away from the cofactor binding site in NS3P/4A relative to NS3P, thereby accommodating NS4A. One result is that catalytic residues His-57 and Asp-81 move closer to Ser-139 and their sidechains adopt more 'traditional' (trypsin-like) orientation. The N-terminus (residues 1-30), while extended in NS3P, is folded into an alpha-helix and beta-strand that cover the bound cofactor of NS3P/4A. A new substrate-binding surface is formed from both the refolded N-terminus and NS4A, potentially affecting substrate residues immediately downstream of the cleavage site. CONCLUSIONS: Direct comparison of the crystal structures of NS3P and NS3P/4A shows that the binding of NS4A improves the anchoring and orientation of the enzyme's catalytic triad. This is consistent with the enhancement of NS3P's weak residual activity upon NS4A binding. There is also significant refolding of the enzyme's N-terminus which provides new interactions with P'-side substrate residues. The binding surface for P'-side substrate residues, including the P1 specificity pocket, changes little after NS4A binding. In summary, we observe a structural basis for improved substrate turnover and affinity that follows complexation of NS3P with its NS4A cofactor.
Subject(s)
Hepacivirus/chemistry , Hepacivirus/enzymology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Enzyme Activation , Humans , Models, Molecular , Protein Binding , Protein Conformation , RNA Helicases , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Structure-Activity RelationshipABSTRACT
Current pharmacological agents for human immunodeficiency virus (HIV) infection include drugs targeted against HIV reverse transcriptase and HIV protease. An understudied therapeutic target is HIV integrase, an essential enzyme that mediates integration of the HIV genome into the host chromosome. The dicaffeoylquinic acids (DCQAs) and the dicaffeoyltartaric acids (DCTAs) have potent activity against HIV integrase in vitro and prevent HIV replication in tissue culture. However, their specificity against HIV integrase in cell culture has been questioned. Thus, the ability of the DCQAs and DCTAs to inhibit binding of HIV type 1 (HIV-1) gp120 to CD4 and their activities against HIV-1 reverse transcriptase and HIV RNase H were studied. The DCQAs and DCTAs inhibited HIV-1 integrase at concentrations between 150 and 840 nM. They inhibited HIV replication at concentrations between 2 and 12 microM. Their activity against reverse transcriptase ranged from 7 microM to greater than 100 microM. Concentrations that inhibited gp120 binding to CD4 exceeded 80 microM. None of the compounds blocked HIV-1 RNase H by 50% at concentrations exceeding 80 microM. Furthermore, when the effects of the DCTAs on reverse transcription in acutely infected cells were measured, they were found to have no activity. Therefore, the DCQAs and DCTAs exhibit > 10- to > 100-fold specificity for HIV integrase, and their activity against integrase in biochemical assays is consistent with their observed anti-HIV activity in tissue culture. Thus, the DCQAs and DCTAs are a potentially important class of HIV inhibitors that act at a site distinct from that of current HIV therapeutic agents.
Subject(s)
Caffeic Acids , Chlorogenic Acid/analogs & derivatives , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , Succinates , Tartrates/pharmacology , Acquired Immunodeficiency Syndrome/drug therapy , Chlorogenic Acid/pharmacology , Cinnamates/pharmacology , HIV Envelope Protein gp120/drug effects , HIV Integrase/drug effects , HIV Integrase/metabolism , HIV Reverse Transcriptase/drug effects , HIV-1/enzymology , HumansABSTRACT
NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related.
Subject(s)
Hepacivirus/enzymology , Serine Endopeptidases/chemistry , Viral Nonstructural Proteins/chemistry , Zinc/chemistry , Binding Sites , Cations, Divalent , Humans , Metals , Protein Processing, Post-Translational , RNA Viruses/enzymology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
Current research indicates that the nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) interacts with a variety of RNA substrates during the progression of the viral life cycle. The RNA features specifically recognized by the protein, however, have yet to be identified. SELEX was used to generate a set of RNAs whose affinities for nucleocapsid were on the order of 2 x 10(-9) M. Comparative analysis revealed that each RNA contains a highly conserved fourteen nucleotide sequence-block. Computer modeling and structure probing experiments indicate that the RNA ligands use the consensus sequence to fold into hairpins with an identical asymmetric bulge. The presence of the nucleocapsid protein protects the asymmetric bulge from ribonuclease attack, suggesting that it is the key element in protein recognition. A search for similar structural motifs within the HIV genome reveals several potential interaction sites for the nucleocapsid protein.
Subject(s)
Capsid Proteins , Capsid/metabolism , Gene Products, gag/metabolism , HIV-1/metabolism , RNA/metabolism , Viral Proteins , Base Sequence , Binding Sites , HIV-1/genetics , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Sequence Analysis , gag Gene Products, Human Immunodeficiency VirusABSTRACT
During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.
Subject(s)
Hepatitis C/enzymology , Viral Nonstructural Proteins/ultrastructure , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Metalloproteins/ultrastructure , Models, Molecular , Molecular Sequence Data , Recombinant Proteins , Sequence Alignment , Trypsin , ZincSubject(s)
Ribonuclease H/analysis , Animals , Base Sequence , Biotin , DNA Probes , Digoxigenin , Immunoenzyme Techniques , Mice , Molecular Sequence Data , Nucleic Acid Heteroduplexes , RNA ProbesSubject(s)
DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Nucleic Acid Conformation , Protein Structure, Secondary , Templates, Genetic , Binding Sites , DNA, Viral/chemistry , DNA, Viral/metabolism , HIV Reverse Transcriptase , Humans , Models, Structural , RNA, Viral/chemistry , RNA, Viral/metabolism , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/metabolismABSTRACT
The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of KmdTTP, Km(dT)16, and kcat for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure. Mutation of Asp256 to alanine abolished the enzymatic activity of pol beta. Conservative replacement with glutamic acid (D256E) led to a 320-fold reduction of kcat compared to wild type. Replacement of Arg254 with an alanine (R254A) resulted in a 50-fold reduction of kcat compared to wild type. The Km(dT)16 of D256E and R254A increased about 18-fold relative to wild type. Replacement of Arg254 with a lysine resulted in a 15-fold decrease in kcat, and a 5-fold increase in the Km(dT)16. These kinetic observations support a role of Asp256 and Arg254 in the positioning of divalent metal ions and substrates in precise geometrical orientation for efficient catalysis. The mutation of Arg258 to alanine (R258A) resulted in a 10-fold increase in KmdTTP and a 65-fold increase in Km(dT)16 but resulted in no change of kcat. These observations are discussed in the context of the three-dimensional structures of the catalytic domain of pol beta and the ternary complex of pol beta, ddCTP, and DNA.
Subject(s)
Arginine , Aspartic Acid , DNA Polymerase I/chemistry , DNA Polymerase I/metabolism , Protein Structure, Secondary , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Brain/enzymology , Cloning, Molecular , DNA Polymerase I/isolation & purification , DNA Primers , DNA, Complementary , Electrophoresis, Polyacrylamide Gel , Gene Library , Humans , Kinetics , Male , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Testis/enzymologyABSTRACT
The reverse transcriptase (RT) of HIV-1 is a plausible target for therapeutic agents aimed at inhibiting propagation of the virus. We have used "irrational drug design", that is, combinatorial chemistry with oligonucleotide libraries, to identify high-affinity ligands aimed at HIV-1 RT. The methodology, termed SELEX (systematic evolution of ligands by exponential enrichment), was employed with a single-stranded DNA library. The selected ssDNA ligands bind HIV-1 RT with Kd values as low as 1 nM and inhibit the RNA-dependent DNA-polymerase activity of the enzyme with Ki values as low as 0.3 nM. We also demonstrate the high specificity of one ligand able to selectively discriminate between the reverse transcriptases of HIV-1, AMV, and MMLV. These ssDNA molecules may be useful as inhibitors or as models for the design of small molecule inhibitors of HIV-1 RT in vivo.
Subject(s)
Antiviral Agents/pharmacology , DNA, Single-Stranded/metabolism , HIV-1/enzymology , Oligodeoxyribonucleotides/pharmacology , Reverse Transcriptase Inhibitors , Antiviral Agents/chemical synthesis , Avian Myeloblastosis Virus/enzymology , Base Sequence , Binding Sites , Cloning, Molecular , DNA, Single-Stranded/chemistry , Databases, Factual , Drug Design , HIV Reverse Transcriptase , Kinetics , Mammary Tumor Virus, Mouse/enzymology , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Polymerase Chain Reaction , Recombinant Proteins/antagonists & inhibitors , Templates, GeneticABSTRACT
We had previously used in vitro RNA selection techniques to describe a consensus RNA pseudoknot that binds and inhibits HIV-1 reverse transcriptase (HIV-RT). In this work we constructed variants of this consensus pseudoknot in order to evaluate the contributions of individual nucleotide identities and secondary structure to affinity for HIV-RT. We have also used chemical modification of ligand RNAs to corroborate the predicted structure of the pseudoknot, to discover which modifiable groups are protected from chemical attack when bound to HIV-RT, and to find which modifications interfere with binding to HIV-RT. A novel interference study is presented which involves selection of ligands from a pool created by mixed reagent oligonucleotide synthesis in order to rapidly determine allowed substitutions of 2'-OCH3 groups for the usual 2'-OH group in such RNA ligands.
Subject(s)
Antiviral Agents/chemistry , RNA/pharmacology , Reverse Transcriptase Inhibitors , Base Sequence , Binding Sites , Consensus Sequence , HIV Reverse Transcriptase , HIV-1/enzymology , Ligands , Molecular Sequence Data , Nucleic Acid Conformation , RNA/chemistry , RNA-Directed DNA Polymerase/metabolism , Structure-Activity RelationshipABSTRACT
The human colon epithelial line HT29 represents a semipermisive cellular system for human immunodeficiency virus type 1 (HIV-1). It could be productively infected with HIV-1 NDK, a Zairian virus isolate highly cytopathic for CD4 positive lymphocytes, whereas infection with the prototype virus HIV-1 LAV was nonproductive. Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control, step of virus/cell cycle, and molecular mechanism responsible for productive versus nonproductive infection of intestinal cells. Both parental viruses and all recombinants retrotranscribed their genomes with a similar kinetics and were able to complete HIV-1 DNA synthesis, HIV-1 LAV provirus present in preintegration complexes could be rescued by cocultivation with T-lymphocytes. However, it was aborted during prolonged cultivation of HT29 cells. Our results suggest that (i) gag/pol region of HIV-1 genome (fragment BssHII255-EcoRI4183) genetically controlled productive infection of intestinal cells and that (ii) the difference between productive and abortive infection occurred before synthesis of HIV-1 mRNA, at the integration level.
Subject(s)
Colon/virology , Genes, gag/genetics , Genes, pol/genetics , HIV-1/physiology , Virus Replication/genetics , Cell Line , Colon/cytology , DNA, Viral/biosynthesis , Gene Products, gag/genetics , Genome, Viral , HIV Reverse Transcriptase , HIV-1/genetics , HIV-1/pathogenicity , Humans , Kinetics , Promoter Regions, Genetic/genetics , RNA-Directed DNA Polymerase/metabolism , Recombinant Fusion Proteins/biosynthesis , Ribonuclease H/metabolism , gag Gene Products, Human Immunodeficiency VirusABSTRACT
DNA and RNA polymerases are enzymes that are primarily responsible for copying genetic material in all living systems. The four polymerases whose structures have been determined by X-ray crystallographic methods have significant similarities at the polymerase active site that are indicative of common requirements for polynucleotide synthesis. Structural studies of complexes of the Klenow fragment of Escherichia coli DNA polymerase I, HIV type 1 reverse transcriptase, and rat DNA polymerase beta with DNA are leading to generalized models for catalysis.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , Nucleic Acids/chemistry , Nucleic Acids/metabolism , Protein Conformation , Animals , DNA Polymerase I/chemistry , DNA Polymerase I/metabolism , Humans , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/metabolism , RatsABSTRACT
The Spo0A transcription factor is responsible for the initiation of sporulation and is active in transcription only after phosphorylation by a specific signal transduction pathway, the phosphorelay. The effect of phosphorylation on the physical properties of Spo0A was determined. Spo0A and Spo0A approximately P both behaved as monomers during Sephacryl chromatography and gel electrophoresis, suggesting that phosphorylation did not modify the oligomerization state of the protein. Trypsin digested Spo0A at a single cleavage site between residues 142 and 143 within a hinge connecting two tightly folded domains. The amino domain retains ability to be phosphorylated by the phosphorelay. The carboxyl domain is active as a DNA-binding protein and retains the sequence specificity of the intact molecule for 0A boxes on the abrB promoter as revealed by footprinting studies. The carboxyl domain stimulated in vitro transcription from the spoIIG promoter 5-fold greater than an equal amount of Spo0A and about half as well as equivalent amounts of Spo0A approximately P. Thus, the unphosphorylated amino domain inhibits the transcription stimulation activity of the carboxyl domain. We suggest that phosphorylation activates transcription regulation functions of Spo0A by modifying the spatial relationships of the amino and carboxyl domains.