DNA Base Pair Mismatches Research Articles

7-Deazapurine and Pyrimidine Nucleoside and Oligonucleotide Cycloadducts Formed by Inverse Diels-Alder Reactions with 3,6-Di(pyrid-2-yl)-1,2,4,5-tetrazine: Ethynylated and Vinylated Nucleobases for Functionalization and Impact of Pyridazine Adducts on DNA Base Pair Stability and Mismatch Discrimination.

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

Analysis of Recurrent DNA Base Pair Mismatches in Eye Cancer Patient Genomes

This research investigates potential genetic drivers for uveal melanoma (UM), the most frequent primary cancer of the eye, through a frequency and ontology analysis of recurrent genetic mutations in UM patients. Genome data of 32 patients, 23 primary and 9 metastatic, was acquired from the U.S. National Institute of Health (NIH) and obtained from samples that were surgically collected from eye enucleations or resected from liver metastases. It was hypothesized that chromosomes 15 and 16 would predominantly exhibit genetic alterations linked to UM due to their role in the expression of eye color. During analysis, DNA from cancerous tumor cells was compared to a reference DNA sequence (extracted from normal tissue) to identify nucleotide base pair mismatches. The locus of each mutation noted to determine what genes mutated. Pareto analysis of cross-patient data performed to identify chromosomes with the most genetic mutations along with any recurrent genetic mutations across patients. A gene ontology (GO) analysis conducted to study the functions of mutated genes and investigate possible links to cancer, such as anomalies in genes with a known role in tumor suppression. A total of 130 genetic mutations were identified (7 recurrent and 123 non-recurrent), with most mutations occurring in chromosomes 3 and X. Recurrent mutations varied from 8.7% to 17.39% occurrence in the UM patient sample. The recurring mutations observed as missense mutations in seven genes. These findings suggest that UM is a recessive heterogeneous disease with selected homozygous mutations. Notably, this study has potential wider significance because the seven genes targeted by recurrent mutations affected in other cancers. It concluded that immunotherapy is a highly promising treatment for Uveal Melanoma due to the disease’s heterogeneous nature

Acetylation of MLH1 by CBP increases cellular DNA mismatch repair activity.

The DNA mismatch repair (MMR) proteins recognize and repair DNA base pair mismatches and insertions/deletions of DNA that have occurred during DNA replication. Additionally, they are involved in regulation of the DNA damage response, including cell cycle checkpoints and apoptosis. Therefore, regulation of these proteins is essential for maintaining genomic integrity. It has been recognized that post-translational modifications, such as phosphorylation, ubiquitination, and acetylation, are being used as an important means to regulate the functions and stability of MMR proteins. Here, we report that a histone acetyltransferase CREB binding protein (CBP) interacts with and acetylates MLH1, a component of the MutLα complex (MLH1-PMS2). Moreover, CBP stabilizes MLH1 by preventing it from degradation via the ubiquitin-proteasome degradation pathway. Consistently, acetylation induced by a pan-histone deacetylase inhibitor, Trichostatin A, promotes the assembly between the MutSα (MSH2-MSH6) and MutLα complexes. Furthermore, overexpression of CBP enhances MMR activities in cells. Overall, our results suggest a novel role of CBP in prolonging MLH1 stability and enhancing MutSα-MutLα complex formation, leading to increased cellular MMR activity.

Structure and dynamics of off-target effects in CRISPR-Cas9.

The occurrence of off-target mutations in CRISPR-Cas9 can limit the applicability of this technology in vivo. At the molecular level, off-target effects are the unselective cleavage of DNA sequences that do not fully match the guide RNA in Cas9. Using enhanced simulation methods, we probed the effect of DNA base pair mismatches in the dynamics of CRISPR-Cas9. We found that, depending on their position and nature, base pair mismatches can induce an opening of the RNA:DNA hybrid, which results in newly formed interactions with the catalytic HNH domain. These interactions can “lock” the HNH domain in an inactive state, exerting a conformational control. These findings were corroborated by several X-ray structures of Cas9 bound to off-target substrates. Based on this success, we used molecular simulations to predict the effect of mismatches at positions that were not captured in the crystallographic structures, providing a more complete overview of the structural and dynamic effects of DNA mismatches in Cas9. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the rational design of guide RNAs and off-target prediction algorithms.

Synthetic Lethality Targeting the Aid/RAD51 Axis for Treatment of AICDA-Expressing Lymphoma

Activation Induced Cytidine Deaminase (AICDA or AID) is a DNA directed cytidine deaminase that induces somatic hypermutation and immunoglobulin class switching in stimulated B-lymphocytes. AICDA is a DNA damaging enzyme, producing DNA base pair mismatches that can subsequently be converted into mutations, DNA single strand breaks (SSB), or DNA double strand breaks (DSB). Although AICDA is normally expressed at high levels only in activated B-cells, a wide range of cancer types show constitutive overexpression of AICDA. In malignant cells, as in B-cells, AICDA promotes hypermutation and genomic instability. We previously demonstrated that cells expressing AICDA are critically dependent on the DNA repair factor RAD51 for their survival. We have developed novel small molecule inhibitors of RAD51 that selectively kill AICDA overexpressing cells. One of these, CYT01A, depletes the nucleus of RAD51, thus reducing DNA double strand break repair activity. We have shown CYT01A to be a potent (EC50~150nM) RAD51 inhibitor that reduces the efficiency of RAD51 focus formation, leading to sensitization to excess DNA damage while preserving baseline recombination/repair activity. Here we present the in vitro and in vivo pharmacologic characterization of CYT01A. We first analyzed potential off-target liabilities by carrying out kinome inhibition and safety panel (Panlabs) screens. CYT01A showed negligible inhibitory activity against 429 kinases, receptors, or other pharmacologically important non-RAD51 targets. These data are consistent with high specificity for AICDA and predict in vivo tolerability. We next measured metabolic stability of CYT01A in mouse liver microsome incubation assays, demonstrating CYT01A stability in both the absence and presence of NADPH. Measurement of Caco-2 cell permeability, as a predictor of oral bioavailability, revealed low to moderate efflux of CYT01A. Consistent with these permeability data, CYT01A showed modest oral availability, but relatively long clearance times. Daily oral dosing at 80 mg/kg for 7-14 days resulted in significant reduction in AICDA expressing lymphomas, in a human-to-mouse xenograft model. This demonstrates efficacious iin vivo exposure despite relatively low oral availability. By contrast, AICDA null lymphomas showed no significant tumor response versus vehicle controls, consistent with the synthetic lethal relationship between AICDA and RAD51. Finally, we demonstrated that repeat daily administration of CYT01A up to the maximum dose tested (80mg/kg) was well tolerated and resulted in no observable changes in complete blood counts (CBC), bone marrow function or output, or body weights. Taken together these data represent a definitive proof-of-principle demonstration that in vivo targeting of RAD51 is an effective strategy to treat AICDA expressing cancers. These data form the basis for continued preclinical development of an AICDA/RAD51 synthetic lethal therapeutic paradigm that may be applicable to a wide range of both solid tumors and hematologic malignancies. DisclosuresMills:Cyteir Therapeutics: Employment, Equity Ownership. Cyr:Cyteir Therapeutics: Employment, Equity Ownership. Maclay:Cyteir Therapeutics: Employment, Equity Ownership. Day:Cyteir Therapeutics: Employment, Equity Ownership. Patrick:Cyteir Therapeutics: Consultancy, Equity Ownership. Clas:Cyteir Therapeutics: Consultancy.

A Small Molecule RAD51 Inhibitor Preferentially Affects Cells Expressing High Cytidine Deaminase Activity

RAD51 is a homologous recombination factor critical for DNA replication, recombination, and DNA double strand break repair. RAD51 forms nucleoprotein filaments at damaged DNA sites or corrupted DNA replication forks to mediate DNA strand exchange, culminating in recombinational repair of breaks and replication fork restart. RAD51 is essential for the repair of DNA lesions induced by agents that stall or collapse DNA replication forks. In this context, RAD51 is an important factor in resistance to DNA replication stress induced by the cellular cytidine deaminase AICDA (also known as AID). AICDA is a DNA-directed cytidine deaminase that normally acts to initiate somatic hypermutation and immunoglobulin class switching in activated B-lymphocytes. However, AICDA is often constitutively overexpressed in tumor cells in a range of cancer types. In transformed cells the cytidine deamination activity of AICDA, and the resulting DNA base pair mismatches, cause tumor cell hypermutation. As a byproduct of its mutational activity, AICDA also destabilizes DNA replication, causing a high concentration of DNA double stranded breaks in AIDCA-expressing cells. This leads to an obligate dependency on RAD51 for DSB repair and cell survival. We previously demonstrated that RAD51 depletion induces acute sensitivity to AICDA activity in transformed cells. We now describe the mechanistic characterization of a small molecule RAD51 inhibitor - CYT01A- that is preferentially cytotoxic to AICDA expressing cells. CYT01A is a potent RAD51 inhibitor (EC50 ~150nM) that promotes DNA replication catastrophe selectively in cells expressing AICDA. Exposure to CYT01A in cell culture leads to the depletion of nuclear RAD51, resulting in diminished nuclear foci formation in response to DNA damage. We demonstrate a dose-dependent cytotoxic response to CYT01A that was reflected by RAD51 foci suppression. We further demonstrate compromised RAD51 nucleoprotein filament formation, evidenced by persistence of Replication Protein A (RPA) foci following CYT01A treatment. RAD51 depletion and reduced foci formation correlates with a reduction in homologous recombination activity. Finally, we show that CYT01A treated cells undergo replication catastrophe, associated with caspase3/7 activation. Collectively, these data indicate that CYT01A prevents RAD51 transport into the nucleus and thereby inhibits DNA repair activity. This demonstrates the feasibility of targeting RAD51 to induce cell death selectively in cells with high DNA replication stress, such as AICDA overexpressing tumor cells. These findings form the basis for a new synthetic lethal therapeutic approach, targeting the AICDA/RAD51 axis in cancers that exhibit high levels of AICDA activity DisclosuresMills:Cyteir Therapeutics: Employment, Equity Ownership. Cyr:Cyteir Therapeutics: Employment, Equity Ownership. Maclay:Cyteir Therapeutics: Employment, Equity Ownership. Day:Cyteir Therapeutics: Employment, Equity Ownership. Khalil:Cyteir Therapeutics: Consultancy, Equity Ownership.

Spontaneous and frequent conformational dynamics induced by A\u2026A mismatch in d(CAA)\xb7d(TAG) duplex

Base pair mismatches in DNA can erroneously be incorporated during replication, recombination, etc. Here, the influence of A…A mismatch in the context of 5′CAA·5′TAG sequence is explored using molecular dynamics (MD) simulation, umbrella sampling MD, circular dichroism (CD), microscale thermophoresis (MST) and NMR techniques. MD simulations reveal that the A…A mismatch experiences several transient events such as base flipping, base extrusion, etc. facilitating B–Z junction formation. A…A mismatch may assume such conformational transitions to circumvent the effect of nonisostericity with the flanking canonical base pairs so as to get accommodated in the DNA. CD and 1D proton NMR experiments further reveal that the extent of B–Z junction increases when the number of A…A mismatch in d(CAA)·d(T(A/T)G) increases (1–5). CD titration studies of d(CAA)·d(TAG)n=5 with the hZαADAR1 show the passive binding between the two, wherein, the binding of protein commences with B–Z junction recognition. Umbrella sampling simulation indicates that the mismatch samples anti…+ syn/+ syn…anti, anti…anti & + syn…+ syn glycosyl conformations. The concomitant spontaneous transitions are: a variety of hydrogen bonding patterns, stacking and minor or major groove extrahelical movements (with and without the engagement of hydrogen bonds) involving the mismatch adenines. These transitions frequently happen in anti…anti conformational region compared with the other three regions as revealed from the lifetime of these states. Further, 2D-NOESY experiments indicate that the number of cross-peaks diminishes with the increasing number of A…A mismatches implicating its dynamic nature. The spontaneous extrahelical movement seen in A…A mismatch may be a key pre-trapping event in the mismatch repair due to the accessibility of the base(s) to the sophisticated mismatch repair machinery.

Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9.

CRISPR-Cas9 is the forefront technology for editing the genome. In this system, the Cas9 protein is programmed with guide RNAs to process DNA sequences that match the guide RNA forming an RNA:DNA hybrid structure. However, the binding of DNA sequences that do not fully match the guide RNA can limit the applicability of CRISPR-Cas9 for genome editing, resulting in the so-called off-target effects. Here, molecular dynamics is used to probe the effect of DNA base pair mismatches within the RNA:DNA hybrid in CRISPR-Cas9. Molecular simulations revealed that the presence of mismatched pairs in the DNA at distal sites with respect to the Protospacer Adjacent Motif (PAM) recognition sequence induces an extended opening of the RNA:DNA hybrid, leading to novel interactions established by the unwound nucleic acids and the protein counterpart. On the contrary, mismatched pairs upstream of the RNA:DNA hybrid are rapidly incorporated within the heteroduplex, with minor effect on the protein-nucleic acid interactions. As a result, mismatched pairs at PAM distal ends interfere with the activation of the catalytic HNH domain, while mismatches fully embedded in the RNA:DNA do not affect the HNH dynamics and enable its activation to cleave the DNA. These findings provide a mechanistic understanding to the intriguing experimental evidence that PAM distal mismatches hamper a proper function of HNH, explaining also why mismatches within the heteroduplex are much more tolerated. This constitutes a step forward in understanding off-target effects in CRISPR-Cas9, which encourages novel structure-based engineering efforts aimed at preventing the onset of off-target effects.

An 111In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells.

Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics. Herein we report the synthesis of a mismatch DNA binding bis-ruthenium(ii) dipyridophenazine (dppz) complex that can be radiolabelled with the Auger electron emitting radionuclide indium-111 (111In). Greater stabilisation accompanied by enhanced MLCT (metal to ligand charge-transfer) luminescence of both the bis-Ru(dppz) chelator and non-radioactive indium-loaded complex was observed in the presence of a TT mismatch-containing duplex compared to matched DNA. The radioactive construct [111In]In-bisRu(dppz) ([111In][In-2]4+) targets cell nuclei and is radiotoxic towards MMR-deficient human colorectal cancer cells showing substantially less detrimental effects in a paired cell line with restored MMR function. Additional cell line studies revealed that [111In][In-2]4+ is preferentially radiotoxic towards MMR-deficient colorectal cancer cells accompanied by increased DNA damage due to 111In decay. The biodistribution of [111In][In-2]4+ in live mice was demonstrated using SPECT. These results illustrate how a Ru(ii) polypyridyl complex can incorporate mismatch DNA binding and radiometal chelation in a single molecule, generating a DNA-targeting AE radiopharmaceutical that displays selective radiotoxicity towards MMR-deficient cancer cells and is compatible with whole organism SPECT imaging.

FeII4L4 Tetrahedron Binds to Nonpaired DNA Bases.

A water-soluble self-assembled supramolecular FeII4L4 tetrahedron binds to single stranded DNA, mismatched DNA base pairs, and three-way DNA junctions. Binding of the coordination cage quenches fluorescent labels on the DNA strand, which provides an optical means to detect the interaction and allows the position of the binding site to be gauged with respect to the fluorescent label. Utilizing the quenching and binding properties of the coordination cage, we developed a simple and rapid detection method based on fluorescence quenching to detect unpaired bases in double-stranded DNA.

Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics.

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations—using Gaussian accelerated MD (GaMD)—are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to “lock” the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to “lock” the catalytic HNH domain in an inactive “conformational checkpoint” is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Molecular Mechanism of Off-Target Effects in CRISPR-Cas9

Author(s): Ricci, Clarisse; Chen, Janice; Miao, Yinglong; Jinek, Martin; Doudna, Jennifer; McCammon, Andrew; Palermo, Giulia | Abstract: Abstract CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Cellular Target of a Rhodium Metalloinsertor is the DNA Base Pair Mismatch.

Defects in DNA mismatch repair (MMR) are commonly found in various cancers, especially in colorectal cancers. Despite the high prevalence of MMR-deficient cancers, mismatch-targeted therapeutics are limited and diagnostic tools are indirect. Here, we examine the cytotoxic properties of a rhodium metalloinsertor, [Rh(phen)(chrysi)(PPO)]2+ (RhPPO) in 27 diverse colorectal cancer cell lines. Despite the low frequency of genomic mismatches and the non-covalent nature of the RhPPO-DNA lesion, RhPPO is on average five times more potent than cisplatin. Importantly, the biological target and profile for RhPPO differs from that of cisplatin. A fluorescent metalloinsertor, RhCy3, was used to demonstrate that the cellular target of RhPPO is the DNA mismatch. RhCy3 represents a direct probe for MMR-deficiency and correlates directly with the cytotoxicity of RhPPO across different cell lines. Overall, our studies clearly indicate that RhPPO and RhCy3 are promising anticancer and diagnostic probes for MMR-deficient cancers, respectively.

DNA Base Pair Mismatches Induce Structural Changes and Alter the Free-Energy Landscape of Base Flip

Double-stranded DNA may contain mismatched base pairs beyond the Watson-Crick pairs, guanine-cytosine and adenine-thymine. Such mismatches bear adverse consequences for human health. We utilize molecular dynamics and metadynamics computer simulations to study the structure and dynamics of both matched and mismatched base pairs. We discover significant differences between the matched and mismatched pairs in structure and base flip work profiles. Mismatched pairs shift more in the plane normal to the DNA strand and exhibit more noncanonical structures, including the e-motif. We discuss the potential implications on the mismatch repair enzymes' detection of DNA mismatches.

338 - Mitochondrial DNA damage in diabetic retinopathy: role of epigenetics

Purpose Diabetes dysfunctions retinal mitochondria and increases base pair mismatches in mitochondrial DNA (mtDNA), and damaged mtDNA impairs the electron transport chain system. A mismatch repair system actively repairs the uncomplimentary base pairs, but in diabetes, the enzyme responsible for cutting the base-mismatches, MutL homolog 1 (Mlh1), is also compromised, which further adds to the damage of mtDNA. In diabetes, retinal DNA methylation machinery is also altered, and the activity of DNA methyl transferases (Dnmts) is significantly increased. Since Mlh1 promoter has over 50 CpG sites, making it a good target for DNA methylation, our aim was to investigate the role of DNA methylation in mtDNA damage in the development of diabetic retinopathy. Methods Retinal microvessels from streptozotocin-induced diabetic mice, with or without Dnmt inhibitor 5-azacytidine (Aza, 2.5mg/kg, intraperitoneal), were analyzed for methylated cytosine (5mC) levels using immuno-capture method and Mlh1 promoter- specific primers. Base-mismatch was quantified by digesting mtDNA amplicons using mismatch specific surveyor endonuclease, followed by DNA fragmentation analysis. Similar experiments were performed in retinal microvessels prepared from human donors with documented diabetic retinopathy. Results Compared to normal mice, retinal microvessels from diabetic mice had decreased Mlh1 expression and increased Dnmt1 and 5mC levels at Mlh1 promoter. In the same mice, base-mismatches in their mtDNA were also increased. Administration of Aza ameliorated diabetes-induced decrease in Mlh1 expression, and prevented increase in Dnmt1 and DNA methylation of Mlh1 promoter. Similarly, compared to age-matched non diabetic human donors, retinal microvessels from donors with established diabetic retinopathy had decreased levels of Mlh1 and increased levels of Dnmt1 and 5mC at Mlh1 promoter. Conclusions Thus, DNA methylation of Mlh1 promoter has a major role in impaired mtDNA damage. Therapies targeted to halt DNA methylation could have potential to prevent/halt mtDNA damage, and the development of diabetic retinopathy.

Platinum(II) Complexes with Sterically Expansive Tetraarylethylene Ligands as Probes for Mismatched DNA

Deficiencies in DNA mismatch repair (MMR) machinery result in greater incidence of DNA base pair mismatches in many types of cancer cells relative to normal cells. Consequently, luminescent probes capable of signaling the presence of mismatched DNA hold promise as potential cancer diagnostic and therapeutic tools. In this study, a series of cyclometalated platinum(II) complexes with sterically expansive tetraarylethylene ligands were synthesized and examined for selective detection of mismatched DNA. Increased steric bulk of the tetraarylethylene ligands in these complexes was observed to correlate with greater preferential luminescence enhancement in the presence of hairpin DNA oligonucleotides containing a mismatched site compared to well-matched oligonucleotides, with the most effective complex displaying ∼14-fold higher emission upon binding CC mismatched oligonucleotides compared to well-matched oligonucleotides. The results indicate binding to mismatched sites in DNA oligonucleotides occurs through metalloinsertion, and the luminescence response increases as a function of thermodynamic destabilization of the mismatch. Luminescence quenching experiments with Cu(phen)22+ and NaI further indicate mismatch binding from the minor groove, consistent with metalloinsertion. Binding to CC mismatched oligonucleotides was also investigated by isothermal titration calorimetry and UV-melting studies. These results demonstrate the efficacy of tetraarylethylene-based platinum(II) complexes for detection of mismatched DNA and establish a new molecular platform for development of organometallic DNA binding agents.

Abstract 347: Targeting the AICDA/RAD51 axis: A novel gain-of-function synthetic lethal therapy for the treatment of AICDA-expressing cancers

Abstract A key characteristic of many cancers is genomic instability, and is often associated with poor prognosis. While genomic instability can promote tumorigenesis, it also provides a therapeutic opportunity for synthetic lethality. The induction of DNA damage creates stress that necessitates highly active DNA repair as a critical survival system for many transformed cells. Recently, therapeutics that target PARP1 have been approved by the FDA as synthetic lethal therapeutics for cancers with deficiencies in BRCA1/2 and associated pathways. Here we present the early development of a new synthetic lethal therapy that leverages gain-of-function abnormalities to selectively target cancer cells. Activation Induced Cytidine Deaminase (AICDA or AID) is a DNA-directed cytidine deaminase that is normally expressed exclusively and transiently in activated B-lymphocytes, where it plays critical roles in somatic hypermutation and immunoglobulin class switching. AICDA is a DNA damaging enzyme, producing DNA base pair mismatches which can subsequently be converted into mutations, DNA single strand breaks (SSB), or DNA double strand breaks (DSB). Numerous cancers show constitutive overexpression of AICDA, leading to hypermutation, genomic instability, and tumor evolution. We have previously demonstrated that cells expressing AICDA are critically dependent upon the DNA repair factor RAD51. Here we present data illustrating the effectiveness of a novel small molecule, CYT02A, in targeting the AICDA/RAD51 axis. Cell culture assays showed CYT02A to be a potent DNA repair inhibitor that targets RAD51 subcellular localization and filament formation. Although CYT02A was stable when incubated with liver microsomes. Although CYT-02A showed limited oral bioavailability, it was stable in liver microsome assyas assays and the pharmacokinetic profile following intravenous injection revealed dose proportionality and acceptable in vivo exposures. CYT02A was efficacious in a human-to-mouse xenograft model of chronic lymphocytic leukemia (CLL). CYT02A was administered intravenously at a concentration of 50 mg/kg to AID+ CLL xenograft mice daily for up to 9 days. Treated mice showed a significant reduction in CLL burden in the bone marrow compared to vehicle control animals. CYT02A was well tolerated by the animals, with no observable change in behavior or complete blood counts (CBC). Taken together, these data validate a novel gain of function synthetic lethal approach targeting the RAD51/AICDA axis. Preclinical efficacy data support this strategy for the treatment of AICDA-expressing cancers. We continue to build upon this foundation to produce a new therapeutic paradigm that may be effective in a wide range of both hematologic malignancies and solid tumors. Citation Format: Muneer Hasham, Kin-hoe Chow, Tyler Maclay, Amber Cyr, Darryl Patrick, Melinda Day, Kevin D. Mills. Targeting the AICDA/RAD51 axis: A novel gain-of-function synthetic lethal therapy for the treatment of AICDA-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 347.

A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers.

Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base pair mismatches in vitro and kill mismatch repair (MMR)-deficient cells at lower concentrations than their MMR-proficient counterparts. A family of metalloinsertors containing rhodium-oxygen ligand coordination, termed "Rh-O" metalloinsertors, has been prepared and shown to have a significant increase in both overall potency and selectivity toward MMR-deficient cells regardless of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies with the second generation of Rh-O metalloinsertors in which an ancillary ligand is varied in both steric bulk and lipophilicity. These complexes, of the form [Rh(L)(chrysi)(PPO)]2+, all include the O-containing PPO ligand (PPO = 2-(pyridine-2-yl)propan-2-ol) and the aromatic inserting ligand chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L is a phenanthroline or bipyridyl derivative. The Rh-O metalloinsertors in this family all show micromolar binding affinities for a 29-mer DNA hairpin containing a single CC mismatch. The complexes display comparable lipophilic tendencies and p Ka values of 8.1-9.1 for dissociation of an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes containing the phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient cells at nanomolar concentrations. Using mass spectral analyses, it is shown that the complexes are taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an off-target organelle that, when targeted by parent metalloinsertors, can lead to nonselective cytotoxicity. Overall, these Rh-O metalloinsertors have distinct and improved behavior compared to previous generations of parent metalloinsertors, making them ideal candidates for further therapeutic assessment.

Hydrogen‐Bond Strength of CC and GG Pairs Determined by Steric Repulsion: Electrostatics and Charge Transfer Overruled

Theoretical and experimental studies have elucidated the bonding mechanism in hydrogen bonds as an electrostatic interaction, which also exhibits considerable stabilization by charge transfer, polarization, and dispersion interactions. Therefore, these components have been used to rationalize the differences in strength of hydrogen‐bonded systems. A completely new viewpoint is presented, in which the Pauli (steric) repulsion controls the mechanism of hydrogen bonding. Quantum chemical computations on the mismatched DNA base pairs CC and GG (C=cytosine, G=guanine) show that the enhanced stabilization and shorter distance of GG is determined entirely by the difference in the Pauli repulsion, which is significantly less repulsive for GG than for CC. This is the first time that evidence is presented for the Pauli repulsion as decisive factor in relative hydrogen‐bond strengths and lengths.

Detection of base-pair mismatches in DNA using graphene-based nanopore device

We present a unique way to detect base-pair mismatches in DNA, leading to a different epigenetic disorder by the method of nanopore sequencing. Based on a tight-binding formulation of a graphene-based nanopore device, using the Green’s function approach we study the changes in the electronic transport properties of the device as we translocate a double-stranded DNA through the nanopore embedded in a zigzag graphene nanoribbon. In the present work we are not only successful in detecting the usual AT and GC pairs but also a set of possible mismatches in the complementary base pairing.