ABSTRACT
Lithium cobalt oxide is a convenient model material for the vast family of cathode materials with a layered structure and still retains some commercial perspectives for microbatteries and some other applications. In this work, we have used ab initio calculations, x-ray diffraction, Raman spectroscopy, and a theoretical physical model, based on quasi-harmonic approximation with anharmonic contributions of the three-phonon and four-phonon processes, to study a temperature-induced change of Raman spectra for LiCoO2. The obtained values of shift and broadening for Eg and A1g bands can be used for quantitative characterization of temperature change, for example, due to laser-induced heating during Raman spectra measurements. The theoretical analysis of the experimental results lets us conclude that Raman spectra changes for LiCoO2 can be explained by the combination of thermal expansion of the crystal lattice and phonon damping by anharmonic coupling with comparable contributions of the three-phonon and four-phonon processes. The obtained results can be further used to develop Raman-based quality control tools.
ABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family that can downregulate the anticancer effects of the type I topoisomerase (TOP1) inhibitors by hydrolyzing the 3'-phosphodiester bond between DNA and the TOP1 residue Y723 in the critical stalled intermediate that is the foundation of TOP1 inhibitor mechanism of action. Thus, TDP1 antagonists are attractive as potential enhancers of TOP1 inhibitors. However, the open and extended nature of the TOP1-DNA substrate-binding region has made the development of TDP1 inhibitors extremely challenging. In this study, starting from our recently identified small molecule microarray (SMM)-derived TDP1-inhibitory imidazopyridine motif, we employed a click-based oxime protocol to extend the parent platform into the DNA and TOP1 peptide substrate-binding channels. We applied one-pot Groebke-Blackburn-Bienayme multicomponent reactions (GBBRs) to prepare the needed aminooxy-containing substrates. By reacting these precursors with approximately 250 aldehydes in microtiter format, we screened a library of nearly 500 oximes for their TDP1 inhibitory potencies using an in vitro florescence-based catalytic assay. Select hits were structurally explored as their triazole- and ether-based isosteres. We obtained crystal structures of two of the resulting inhibitors bound to the TDP1 catalytic domain. The structures reveal that the inhibitors form hydrogen bonds with the catalytic His-Lys-Asn triads ("HKN" motifs: H263, K265, N283 and H493, K495, N516), while simultaneously extending into both the substrate DNA and TOP1 peptide-binding grooves. This work provides a structural model for developing multivalent TDP1 inhibitors capable of binding in a tridentate fashion with a central component situated within the catalytic pocket and extensions that project into both the DNA and TOP1 peptide substrate-binding regions.
ABSTRACT
Eukaryotic topoisomerases I (TOP1) are ubiquitous enzymes removing DNA torsional stress. However, there is little data concerning the three-dimensional structure of TOP1 in the absence of DNA, nor how the DNA molecule can enter/exit its closed conformation. Here, we solved the structure of thermostable archaeal Caldiarchaeum subterraneum CsTOP1 in an apo-form. The enzyme displays an open conformation resulting from one substantial rotation between the capping (CAP) and the catalytic (CAT) modules. The junction between these two modules is a five-residue loop, the hinge, whose flexibility permits the opening/closing of the enzyme and the entry of DNA. We identified a highly conserved tyrosine near the hinge as mediating the transition from the open to closed conformation upon DNA binding. Directed mutagenesis confirmed the importance of the hinge flexibility, and linked the enzyme dynamics with sensitivity to camptothecin, a TOP1 inhibitor targeting the TOP1 enzyme catalytic site in the closed conformation.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , Camptothecin/pharmacology , Catalytic Domain , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Damage , DNA Repair , DNA Topoisomerases, Type I/genetics , DNA-Binding Proteins , Humans , Models, Molecular , Protein Conformation , Sequence AlignmentABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand-protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.
ABSTRACT
Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5'-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.
Subject(s)
DNA-Binding Proteins/genetics , Flavins/chemical synthesis , Phosphodiesterase Inhibitors/chemical synthesis , Phosphoric Diester Hydrolases/genetics , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Drug Screening Assays, Antitumor , Drug Synergism , Etoposide/pharmacology , Flavins/chemistry , Flavins/pharmacology , Humans , Mitoxantrone/pharmacology , Molecular Docking Simulation , Molecular Structure , Mutation , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacologyABSTRACT
Tyrosyl DNA-phosphodiesterase I (TDP1) repairs type IB topoisomerase (TOP1) cleavage complexes generated by TOP1 inhibitors commonly used as anticancer agents. TDP1 also removes DNA 3' end blocking lesions generated by chain-terminating nucleosides and alkylating agents, and base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors is proposed to synergize with topoisomerase targeting drugs to enhance selectivity against cancer cells exhibiting deficiencies in parallel DNA repair pathways. A crystallographic fragment screening campaign against the catalytic domain of TDP1 was conducted to identify new lead compounds. Crystal structures revealed two fragments that bind to the TDP1 active site and exhibit inhibitory activity against TDP1. These fragments occupy a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, several fragment derivatives have been prepared and evaluated in biochemical assays. These results demonstrate that fragment-based methods can be a highly feasible approach toward the discovery of small-molecule chemical scaffolds to target TDP1, and for the first time, we provide co-crystal structures of small molecule inhibitors bound to TDP1, which could serve for the rational development of medicinal TDP1 inhibitors.
Subject(s)
Enzyme Inhibitors/chemistry , Ligands , Phosphoric Diester Hydrolases/chemistry , Protein Conformation , Base Sequence , Catalytic Domain/genetics , Crystallography , DNA Repair/genetics , Histidine/analogs & derivatives , Histidine/chemistry , Histidine/isolation & purification , Humans , Models, Molecular , Phosphoric Diester Hydrolases/genetics , Signal Transduction , Small Molecule Libraries/chemistryABSTRACT
Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.
Subject(s)
Antineoplastic Agents/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Etoposide/pharmacology , Flavins/pharmacology , Topoisomerase II Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Chickens , Drug Synergism , Flavins/chemical synthesis , Flavins/chemistry , Humans , Mice , Molecular Structure , Phosphoric Diester Hydrolases , Structure-Activity RelationshipABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)-DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC50 value of 7 µM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future efforts for the discovery of natural product-based TOP1 and TDP1 inhibitors.
Subject(s)
DNA Topoisomerases, Type I/metabolism , Drug Design , Phenanthridines/chemical synthesis , Phenanthridines/pharmacology , Phosphoric Diester Hydrolases/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Chemistry Techniques, Synthetic , DNA Cleavage/drug effects , DNA Topoisomerases, Type I/chemistry , Humans , Models, Molecular , Phenanthridines/chemistry , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/chemistry , Protein Conformation , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/pharmacologyABSTRACT
HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69â»75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.
Subject(s)
Gene Products, vpr/chemistry , Gene Products, vpr/metabolism , HIV Infections/virology , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/physiology , Peptides/pharmacology , Amino Acid Sequence , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein MultimerizationABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an ubiquitous DNA repair enzyme present in yeast, plants and animals. It removes a broad range of blocking lesions at the ends of DNA breaks. The catalytic core of TDP1 consists in a pair of conserved histidine-lysine-asparagine (HKN) motifs. Analysis of the human TDP1 (hTDP1) crystal structure reveals potential involvement of additional residues that shape the substrate binding site. In this biochemical study, we analyzed four such conserved residues, tyrosine 204 (Y204), phenylalanine 259 (F259), serine 400 (S400) and tryptophan 590 (W590). We show that the F259 residue of hTDP1 is critical for both 3'- and 5'-phosphodiesterase catalysis. We propose that the double π-π interactions of the F259 residue with the -2 and -3 nucleobases serve to position the nucleopeptide substrate in phase with the active site histidines of hTDP1. Mutating Y204 of hTDP1 to phenylalanine (Y204F), as in fly and yeast TDP1 enzymes, had minor impact on TDP1 activity. In constrast, we find that S400 enhances 3'-processing activity while it suppresses 5'-processing activity, thereby promoting specificity for 3'-substrates. W590 is selectively important for 5'-processing. These results reveal the impact of conserved amino acid residues that participate in defining the DNA binding groove around the dual HKN catalytic core motif of TDP1, and their differential roles in facilitating the 3'- vs 5'-end processing activities of hTDP1.
Subject(s)
Catalytic Domain , DNA Cleavage , DNA Damage , Phosphoric Diester Hydrolases/metabolism , Amino Acid Sequence , Conserved Sequence , DNA/metabolism , DNA Repair , Humans , Phosphoric Diester Hydrolases/chemistry , Sequence AlignmentABSTRACT
Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a recently discovered enzyme specifically repairing topoisomerase II (TOP2)-mediated DNA damage. It has been shown that inhibition of TDP2 synergize with TOP2 inhibitors. Herein, we report the discovery of the furoquinolinedione chemotype as a suitable skeleton for the development of selective TDP2 inhibitors. Compound 1 was identified as a TDP2 inhibitor as a result of screening our in-house compound library for compounds selective for TDP2 vs. TDP1. Further SAR studies provide several selective TDP2 inhibitors at low-micromolar range. The most potent compound 74 shows inhibitory activity with IC50 of 1.9 and 2.1⯵M against recombinant TDP2 and TDP2 in whole cell extracts (WCE), respectively.
Subject(s)
Nuclear Proteins/antagonists & inhibitors , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Transcription Factors/antagonists & inhibitors , Animals , Cell Line , Chickens , DNA-Binding Proteins , Humans , Molecular Docking Simulation , Nuclear Proteins/metabolism , Phosphodiesterase Inhibitors/chemical synthesis , Phosphoric Diester Hydrolases , Quinolines/chemical synthesis , Recombinant Proteins/metabolism , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes resistance to TOP2-targeted cancer therapy. Inhibiting TDP2 could sensitize cancer cells toward TOP2 inhibitors. However, potent TDP2 inhibitors with favorable physicochemical properties are not yet reported. Therefore, there is a need to search for novel molecular scaffolds capable of inhibiting TDP2. We report herein a new simple, robust, homogenous mix-and-read fluorescence biochemical assay based using humanized zebrafish TDP2 (14M_zTDP2), which provides biochemical and molecular structure basis for TDP2 inhibitor discovery. The assay was validated by screening a preselected library of 1600 compounds (Z'â¯≥â¯0.72) in a 384-well format, and by running in parallel gel-based assays with fluorescent DNA substrates. This library was curated via virtual high throughput screening (vHTS) of 460,000 compounds from Chembridge Library, using the crystal structure of the novel surrogate protein 14M_zTDP2. From this primary screening, we selected the best 32 compounds (2% of the library) to further assess their TDP2 inhibition potential, leading to the IC50 determination of 10 compounds. Based on the dose-response curve profile, pan-assay interference compounds (PAINS) structure identification, physicochemical properties and efficiency parameters, two hit compounds, 11a and 19a, were tested using a novel secondary fluorescence gel-based assay. Preliminary structure-activity relationship (SAR) studies identified guanidine derivative 12a as an improved hit with a 6.4-fold increase in potency over the original HTS hit 11a. This study highlights the importance of the development of combination approaches (biochemistry, crystallography and high throughput screening) for the discovery of TDP2 inhibitors.
Subject(s)
High-Throughput Screening Assays , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/metabolism , Animals , Biological Assay , Fluorescence , Phosphodiesterase Inhibitors/chemistry , Phosphoric Diester Hydrolases/chemistry , Small Molecule Libraries , Structure-Activity Relationship , ZebrafishABSTRACT
2'-C-cyano-2'-deoxy-1-ß-d-arabino-pentofuranosylcytosine (CNDAC) is the active metabolite of the anticancer drug, sapacitabine. CNDAC is incorporated into the genome during DNA replication and subsequently undergoes ß-elimination that generates single-strand breaks with abnormal 3'-ends. Because tyrosyl-DNA phosphodiesterase 1 (TDP1) selectively hydrolyzes nonphosphorylated 3'-blocking ends, we tested its role in the repair of CNDAC-induced DNA damage. We show that cells lacking TDP1 (avian TDP1-/- DT40 cells and human TDP1 KO TSCER2 and HCT116 cells) exhibit marked hypersensitivity to CNDAC. We also identified BRCA1, FANCD2, and PCNA in the DNA repair pathways to CNDAC. Comparing CNDAC with the chemically related arabinosyl nucleoside analog, cytosine arabinoside (cytarabine, AraC) and the topoisomerase I inhibitor camptothecin (CPT), which both generate 3'-end blocking DNA lesions that are also repaired by TDP1, we found that inactivation of BRCA2 renders cells hypersensitive to CNDAC and CPT but not to AraC. By contrast, cells lacking PARP1 were only hypersensitive to CPT but not to CNDAC or AraC. Examination of TDP1 expression in the cancer cell line databases (CCLE, GDSC, NCI-60) and human cancers (TCGA) revealed a broad range of expression of TDP1, which was correlated with PARP1 expression, TDP1 gene copy number and promoter methylation. Thus, this study identifies the importance of TDP1 as a novel determinant of response to CNDAC across various cancer types (especially non-small cell lung cancers), and demonstrates the differential involvement of BRCA2, PARP1, and TDP1 in the cellular responses to CNDAC, AraC, and CPT. Mol Cancer Ther; 16(11); 2543-51. ©2017 AACR.
Subject(s)
BRCA2 Protein/genetics , Colorectal Neoplasms/drug therapy , Phosphoric Diester Hydrolases/genetics , Poly (ADP-Ribose) Polymerase-1/genetics , Arabinonucleosides/administration & dosage , Arabinonucleosides/adverse effects , Camptothecin/administration & dosage , Camptothecin/chemistry , Colorectal Neoplasms/genetics , Cytarabine/administration & dosage , Cytarabine/adverse effects , Cytarabine/analogs & derivatives , Cytarabine/chemistry , Cytosine/administration & dosage , Cytosine/adverse effects , Cytosine/analogs & derivatives , DNA Breaks, Single-Stranded/drug effects , DNA Damage/drug effects , DNA Repair/drug effects , DNA Repair/genetics , DNA Replication/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , HumansABSTRACT
The 7-azaindenoisoquinolines are cytotoxic topoisomerase I (Top1) inhibitors. Previously reported representatives bear a 3-nitro group. The present report documents the replacement of the potentially genotoxic 3-nitro group by 3-chloro and 3-fluoro substituents, resulting in compounds with high Top1 inhibitory activities and potent cytotoxicities in human cancer cell cultures and reduced lethality in an animal model. Some of the new Top1 inhibitors also possess moderate inhibitory activities against tyrosyl-DNA phosphodiesterase 1 (TDP1) and tyrosyl-DNA phosphodiesterase 2 (TDP2), two enzymes that are involved in DNA damage repair resulting from Top1 inhibitors, and they produce significantly more DNA damage in cancer cells than in normal cells. Eighteen of the new compounds had cytotoxicity mean-graph midpoint (MGM) GI50 values in the submicromolar (0.033-0.630 µM) range. Compounds 16b and 17b are the most potent in human cancer cell cultures with MGM GI50 values of 0.063 and 0.033 µM, respectively. Possible binding modes to Top1 and TDP1were investigated by molecular modeling.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , Drug Design , Isoquinolines/pharmacology , Topoisomerase I Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , DNA Cleavage , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/chemistry , Tumor Cells, Cultured , ZebrafishABSTRACT
Tdp1 and Tdp2 are two tyrosyl-DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both Tdp1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, Tdp1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, Tdp1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, Tdp1, and Tdp2 was used to design the inhibitors and facilitate the structure-activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA damage in the cancer cells vs normal cells.
Subject(s)
Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/pharmacology , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/pharmacology , Cells, Cultured , Humans , Structure-Activity RelationshipABSTRACT
Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed.
Subject(s)
Catalytic Domain , DNA, Viral/metabolism , Drug Resistance, Viral/drug effects , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , Biocatalysis/drug effects , Guanine/metabolism , HIV Integrase/chemistry , HIV Integrase Inhibitors/chemistry , Ions , Magnesium/pharmacology , Models, Molecular , Mutation/genetics , Substrate Specificity/drug effectsABSTRACT
UDP and UDP-glucose activate the P2Y14 receptor (P2Y14R) to modulate processes related to inflammation, diabetes, and asthma. A computational pipeline suggested alternatives to naphthalene of a previously reported P2Y14R antagonist (3, PPTN) using docking and molecular dynamics simulations on a hP2Y14R homology model based on P2Y12R structures. By reevaluating the binding of 3 to P2Y14R computationally, two alternatives, i.e., alkynyl and triazolyl derivatives, were identified. Improved synthesis of fluorescent antagonist 4 enabled affinity quantification (IC50s, nM) using flow cytometry of P2Y14R-expressing CHO cells. p-F3C-phenyl-triazole 65 (32) was more potent than a corresponding alkyne 11. Thus, additional triazolyl derivatives were prepared, as guided by docking simulations, with nonpolar aryl substituents favored. Although triazoles were less potent than 3 (6), simpler synthesis facilitated further structural optimization. Additionally, relative P2Y14R affinities agreed with predicted binding of alkynyl and triazole analogues. These triazoles, designed through a structure-based approach, can be assessed in disease models.
Subject(s)
Drug Design , Purinergic P2 Receptor Antagonists/chemistry , Purinergic P2 Receptor Antagonists/pharmacology , Receptors, Purinergic P2/metabolism , Triazoles/chemistry , Triazoles/pharmacology , Animals , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , CHO Cells , Cricetulus , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Structure-Activity RelationshipABSTRACT
Tyrosyl-DNA phosphodiesterase 2 repairs irreversible topoisomerase II-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and processes replication intermediates for picornaviruses (VPg unlinkase) and hepatitis B virus. There is currently no TDP2 inhibitor in clinical development. Here, we report a series of deazaflavin derivatives that selectively inhibit the human TDP2 enzyme in a competitive manner both with recombinant and native TDP2. We show that mouse, fish, and C. elegans TDP2 enzymes are highly resistant to the drugs and that key protein residues are responsible for drug resistance. Among them, human residues L313 and T296 confer high resistance when mutated to their mouse counterparts. Moreover, deazaflavin derivatives show potent synergy in combination with the topoisomerase II inhibitor etoposide in human prostate cancer DU145 cells and TDP2-dependent synergy in TK6 human lymphoblast and avian DT40 cells. Deazaflavin derivatives represent the first suitable platform for the development of potent and selective TDP2 inhibitors.
Subject(s)
Flavins/pharmacology , Nuclear Proteins/antagonists & inhibitors , Phosphodiesterase Inhibitors/pharmacology , Transcription Factors/antagonists & inhibitors , Animals , Cell Line , DNA-Binding Proteins , Flavins/chemistry , Humans , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphodiesterase Inhibitors/chemistry , Phosphoric Diester Hydrolases , Point Mutation , Proton Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Envelope Protein gp120/antagonists & inhibitors , HIV Integrase/metabolism , HIV-1/drug effects , HIV-1/enzymology , Mannans/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Dose-Response Relationship, Drug , HIV Integrase Inhibitors/chemical synthesis , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , HIV-1/metabolism , Mannans/chemical synthesis , Mannans/chemistry , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.