ABSTRACT
AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1's redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5-10-fold) with PK studies demonstrating efficacious doses for translation to clinic.
Subject(s)
DNA-(Apurinic or Apyrimidinic Site) Lyase , Neoplasms , Adult , Humans , Animals , Mice , Angiogenesis Inhibitors , Apoptosis , Biological Assay , Neoplasms/drug therapy , Tumor MicroenvironmentABSTRACT
The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.
Subject(s)
DNA-Directed DNA Polymerase , Drug Discovery , DNA-Directed DNA Polymerase/metabolism , Binding Sites , Endonucleases/metabolism , Crystallography, X-Ray , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolismABSTRACT
The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525 was found to contain the binding determinant of the recombinant human angiotensin-converting enzyme 2, with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to recombinant human angiotensin-converting enzyme 2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.
Subject(s)
Angiotensin-Converting Enzyme 2 , Peptide Library , Severe acute respiratory syndrome-related coronavirus , Spike Glycoprotein, Coronavirus , Humans , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Protein Binding , Severe acute respiratory syndrome-related coronavirus/chemistry , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/immunology , Severe acute respiratory syndrome-related coronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
Targeting of the multifunctional enzyme apurinic/apyrimidinic endonuclease I/redox factor 1 (APE1) has produced small molecule inhibitors of both its endonuclease and redox activities. While one of the small molecules, the redox inhibitor APX3330, completed a Phase I clinical trial for solid tumors and a Phase II clinical trial for Diabetic Retinopathy/Diabetic Macular Edema, the mechanism of action for this drug has yet to be fully understood. Here, we demonstrate through HSQC NMR studies that APX3330 induces chemical shift perturbations (CSPs) of both surface and internal residues in a concentration-dependent manner, with a cluster of surface residues defining a small pocket on the opposite face from the endonuclease active site of APE1. Furthermore, APX3330 induces partial unfolding of APE1 as evidenced by a time-dependent loss of chemical shifts for approximately 35% of the residues within APE1 in the HSQC NMR spectrum. Notably, regions that are partially unfolded include adjacent strands within one of two beta sheets that comprise the core of APE1. One of the strands comprises residues near the N-terminal region and a second strand is contributed by the C-terminal region of APE1, which serves as a mitochondrial targeting sequence. These terminal regions converge within the pocket defined by the CSPs. In the presence of a duplex DNA substrate mimic, removal of excess APX3330 resulted in refolding of APE1. Our results are consistent with a reversible mechanism of partial unfolding of APE1 induced by the small molecule inhibitor, APX3330, defining a novel mechanism of inhibition.
ABSTRACT
The integrated stress response (ISR) is an important mechanism by which cells confer protection against environmental stresses. Central to the ISR is a collection of related protein kinases that monitor stress conditions, such as Gcn2 (EIF2AK4) that recognizes nutrient limitations, inducing phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Gcn2 phosphorylation of eIF2 lowers bulk protein synthesis, conserving energy and nutrients, coincident with preferential translation of stress-adaptive gene transcripts, such as that encoding the Atf4 transcriptional regulator. While Gcn2 is central for cell protection to nutrient stress and its depletion in humans leads to pulmonary disorders, Gcn2 can also contribute to the progression of cancers and facilitate neurological disorders during chronic stress. Consequently, specific ATP-competitive inhibitors of Gcn2 protein kinase have been developed. In this study, we report that one such Gcn2 inhibitor, Gcn2iB, can activate Gcn2, and we probe the mechanism by which this activation occurs. Low concentrations of Gcn2iB increase Gcn2 phosphorylation of eIF2 and enhance Atf4 expression and activity. Of importance, Gcn2iB can activate Gcn2 mutants devoid of functional regulatory domains or with certain kinase domain substitutions derived from Gcn2-deficient human patients. Other ATP-competitive inhibitors can also activate Gcn2, although there are differences in their mechanisms of activation. These results provide a cautionary note about the pharmacodynamics of eIF2 kinase inhibitors in therapeutic applications. Compounds designed to be kinase inhibitors that instead directly activate Gcn2, even loss of function variants, may provide tools to alleviate deficiencies in Gcn2 and other regulators of the ISR.
Subject(s)
Eukaryotic Initiation Factor-2 , Protein Serine-Threonine Kinases , Humans , Adenosine Triphosphate/metabolism , Enzyme Activation/drug effects , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host-guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5'-CTTBPPBBSSZZSAAG, 5'-CTTSSPBZPSZBBAAG and 5'-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, and B:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters of P:Z and B:S pairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further, Z can participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.
Subject(s)
DNA , Oligonucleotides , Mice , Animals , DNA/chemistry , Oligonucleotides/chemistryABSTRACT
Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)â¢BLMA2 "green" (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5'-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)â¢BLMA2 into preformed host-guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA2 was found to be similar to those reported earlier at the same DNA site for BLMB2, the intercalated bithiazole of BLMB2 is "flipped" 180Ë relative to DNA-bound BLMA2. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.
Subject(s)
Bleomycin , Ferric Compounds , Bleomycin/chemistry , DNA/chemistry , Binding SitesABSTRACT
Y-encoded transcription factor SRY initiates male differentiation in therian mammals. This factor contains a high-mobility-group (HMG) box, which mediates sequence-specific DNA binding with sharp DNA bending. A companion article in this issue described sex-reversal mutations at box position 72 (residue 127 in human SRY), invariant as Tyr among mammalian orthologs. Although not contacting DNA, the aromatic ring seals the domain's minor wing at a solvent-exposed junction with a basic tail. A seeming paradox was posed by the native-like biochemical properties of inherited Swyer variant Y72F: its near-native gene-regulatory activity is consistent with the father's male development, but at odds with the daughter's XY female somatic phenotype. Surprisingly, aromatic rings (Y72, F72 or W72) confer higher transcriptional activity than do basic or polar side chains generally observed at solvated DNA interfaces (Arg, Lys, His or Gln). Whereas biophysical studies (time-resolved fluorescence resonance energy transfer and heteronuclear NMR spectroscopy) uncovered only subtle perturbations, dissociation of the Y72F complex was markedly accelerated relative to wild-type. Studies of protein-DNA solvation by molecular-dynamics (MD) simulations of an homologous high-resolution crystal structure (SOX18) suggest that Y72 para-OH anchors a network of water molecules at the tail-DNA interface, perturbed in the variant in association with nonlocal conformational fluctuations. Loss of the Y72 anchor among SRY variants presumably "unclamps" its basic tail, leading to (a) rapid DNA dissociation despite native affinity and (b) attenuated transcriptional activity at the edge of sexual ambiguity. Conservation of Y72 suggests that this water-mediated clamp operates generally among SRY and metazoan SOX domains.
Subject(s)
Sex Determination Processes , Transcription Factors , Animals , Female , Humans , Male , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation , Mammals/genetics , Mammals/metabolism , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Sex Determination Processes/genetics , Sex Determination Processes/physiologyABSTRACT
While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,ß-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol.
ABSTRACT
A fundamental property of DNA built from four informational nucleotide units (GCAT) is its ability to adopt different helical forms within the context of the Watson-Crick pair. Well-characterized examples include A-, B-, and Z-DNA. For this study, we created an isoinformational biomimetic polymer, built (like standard DNA) from four informational "letters", but with the building blocks being artificial. This ALternative Isoinformational ENgineered (ALIEN) DNA was hypothesized to support two nucleobase pairs, the P:Z pair matching 2-amino-imidazo-[1,2a]-1,3,5-triazin-[8H]-4-one with 6-amino-3-5-nitro-1H-pyridin-2-one and the B:S pair matching 6-amino-4-hydroxy-5-1H-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one. We report two structures of ALIEN DNA duplexes at 1.2 Å resolution and a third at 1.65 Å. All of these are built from a single self-complementary sequence (5'-CTSZZPBSBSZPPBAG) that includes 12 consecutive ALIEN nucleotides. We characterized the helical, nucleobase pair, and dinucleotide step parameters of ALIEN DNA in these structures. In addition to showing that ALIEN pairs retain basic Watson-Crick pairing geometry, two of the ALIEN DNA structures are characterized as A-form DNA and one as B-form DNA. We identified parameters that map differences effecting the transition between the two helical forms; these same parameters distinguish helical forms of isoinformational natural DNA. Collectively, our analyses suggest that ALIEN DNA retains essential structural features of natural DNA, not only its information density and Watson-Crick pairing but also its ability to adopt two canonical forms.
Subject(s)
DNA, B-Form , DNA , Base Pairing , DNA/chemistry , Nucleic Acid Conformation , Nucleotides/chemistryABSTRACT
Extensive portions of the human genome have unknown function, including those derived from transposable elements. One such element, the DNA transposon Hsmar1, entered the primate lineage approximately 50 million years ago leaving behind terminal inverted repeat (TIR) sequences and a single intact copy of the Hsmar1 transposase, which retains its ancestral TIR-DNA-binding activity, and is fused with a lysine methyltransferase SET domain to constitute the chimeric SETMAR gene. Here, we provide a structural basis for recognition of TIRs by SETMAR and investigate the function of SETMAR through genome-wide approaches. As elucidated in our 2.37 Å crystal structure, SETMAR forms a dimeric complex with each DNA-binding domain bound specifically to TIR-DNA through the formation of 32 hydrogen bonds. We found that SETMAR recognizes primarily TIR sequences (â¼5000 sites) within the human genome as assessed by chromatin immunoprecipitation sequencing analysis. In two SETMAR KO cell lines, we identified 163 shared differentially expressed genes and 233 shared alternative splicing events. Among these genes are several pre-mRNA-splicing factors, transcription factors, and genes associated with neuronal function, and one alternatively spliced primate-specific gene, TMEM14B, which has been identified as a marker for neocortex expansion associated with brain evolution. Taken together, our results suggest a model in which SETMAR impacts differential expression and alternative splicing of genes associated with transcription and neuronal function, potentially through both its TIR-specific DNA-binding and lysine methyltransferase activities, consistent with a role for SETMAR in simian primate development.
Subject(s)
Genome, Human , Histone-Lysine N-Methyltransferase/genetics , Primates/genetics , Animals , Biological Evolution , Brain/metabolism , DNA Transposable Elements/genetics , Genome-Wide Association Study , Histone-Lysine N-Methyltransferase/metabolism , Humans , Inverted Repeat Sequences , Lysine/genetics , Primates/metabolism , Transposases/chemistryABSTRACT
Multicopper oxidases (MCOs) represent a diverse family of enzymes that catalyze the oxidation of either an organic or a metal substrate with concomitant reduction of dioxygen to water. These enzymes contain variable numbers of cupredoxin domains, two, three or six per subunit, and rely on four copper ions, a single type I copper and three additional copper ions organized in a trinuclear cluster (TNC), with one type II and two type III copper ions, to catalyze the reaction. Here, two crystal structures and the enzymatic characterization of Marinithermus hydrothermalis MCO, a two-domain enzyme, are reported. This enzyme decolorizes Congo Red dye at 70°C in the presence of high halide concentrations and may therefore be useful in the detoxification of industrial waste that contains dyes. In two distinct crystal structures, MhMCO forms the trimers seen in other two-domain MCOs, but differs from these enzymes in that four trimers interact to create a dodecamer. This dodecamer of MhMCO forms a closed ball-like structure and has implications for the sequestration of bound divalent metal ions as well as substrate accessibility. In each subunit of the dodecameric structures, a Trp residue, Trp351, located between the type I and TNC sites exists in two distinct conformations, consistent with a potential role in facilitating electron transfer in the enzyme.
Subject(s)
Bacteria/enzymology , Copper/metabolism , Laccase/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Protein MultimerizationABSTRACT
Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with â¼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of â¼2-8% and crystal structures from â¼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).
Subject(s)
Drug Discovery , Pharmaceutical Preparations , Crystallography, X-Ray , DNA Damage , DNA RepairABSTRACT
DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.
Subject(s)
DNA Replication , DNA/biosynthesis , Protein Engineering , Taq Polymerase , DNA/genetics , Taq Polymerase/chemistry , Taq Polymerase/geneticsABSTRACT
Obtaining semisynthetic microorganisms that exploit the information density of "hachimoji" DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the "hachimoji" P:Z nucleobase pair with a similar efficiency to that seen for Watson-Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the "hachimoji" Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA/chemistry , Base Pairing , DNA/metabolism , DNA-Directed DNA Polymerase/metabolism , Models, MolecularABSTRACT
We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.
Subject(s)
Base Pairing , DNA/chemistry , DNA/genetics , Nucleotides/chemistry , RNA/chemistry , RNA/genetics , Crystallography , Fluorescence , Nucleic Acid Conformation , Polyelectrolytes/chemistry , Synthetic Biology , ThermodynamicsABSTRACT
Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential base excision repair enzyme that is upregulated in a number of cancers, contributes to resistance of tumors treated with DNA-alkylating or -oxidizing agents, and has recently been identified as an important therapeutic target. In this work, we identified hot spots for binding of small organic molecules experimentally in high resolution crystal structures of APE1 and computationally through the use of FTMAP analysis ( http://ftmap.bu.edu/ ). Guided by these hot spots, a library of drug-like macrocycles was docked and then screened for inhibition of APE1 endonuclease activity. In an iterative process, hot-spot-guided docking, characterization of inhibition of APE1 endonuclease, and cytotoxicity of cancer cells were used to design next generation macrocycles. To assess target selectivity in cells, selected macrocycles were analyzed for modulation of DNA damage. Taken together, our studies suggest that macrocycles represent a promising class of compounds for inhibition of APE1 in cancer cells.
Subject(s)
DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Lactams, Macrocyclic/pharmacology , Lactones/pharmacology , Catalytic Domain , Cell Line, Tumor , DNA Damage/drug effects , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Humans , Lactams, Macrocyclic/chemical synthesis , Lactams, Macrocyclic/metabolism , Lactones/chemical synthesis , Lactones/metabolism , Molecular Docking Simulation , Molecular Structure , Protein Binding , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that appear to support molecular recognition with the same proficiency as standard Watson-Crick DNA. However, these both violate size complementarity (big pairs with small), retaining hydrogen bond complementarity (donors pair with acceptors) as their only specificity principle. They exclude mismatches as well as standard Watson-Crick DNA excludes mismatches. In crystal structures, these "skinny" and "fat" systems form the expected hydrogen bonds, while conferring novel minor groove properties to the resultant duplex regions of the DNA oligonucleotides. Further, computational tools, previously tested primarily on natural DNA, appear to work well for these two new molecular recognition systems, offering a validation of the power of modern computational biology. These new molecular recognition systems may have application in materials science and synthetic biology, and in developing our understanding of alternative ways that genetic information might be stored and transmitted.
Subject(s)
DNA/chemistry , Base Pairing , Models, Molecular , Nucleic Acid ConformationABSTRACT
The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-d-2'- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/chemistry , Base Pairing/genetics , DNA-Directed DNA Polymerase/chemistry , Nucleic Acid Conformation , Nucleotides/chemistry , Amino Acid Substitution/genetics , Catalytic Domain/genetics , Crystallography, X-RayABSTRACT
A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson-Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host-guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs.