Helicase Activity Research Articles

Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment

Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying CsfcrRNA complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the CsfcrRNA complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.

Monitoring RNA restructuring in a human cell-free extract reveals eIF4A-dependent and eIF4A-independent unwinding activity

The canonical DEAD-box helicase, eukaryotic initiation factor (eIF) 4A, unwinds 5' UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to monitor helicase activity precisely in the 5' UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5' UTR-dependent duplex unwinding in the absence or presence of an eIF4A inhibitor (hippuristanol), a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that the duplex unwinding activity in the cell-free extract is roughly evenly split between eIF4A-dependent and eIF4A-independent mechanisms. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.

Biochemical properties of naturally occurring human bloom helicase variants.

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

Accurate sequencing of DNA motifs able to form alternative (non-B) structures.

Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases. Because sequencing technologies use these enzymes, they might possess increased errors at non-B structures. To evaluate this, we analyzed error rates, read depth, and base quality of Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT) sequencing at non-B motifs. All technologies showed altered sequencing success for most non-B motif types, although this could be owing to several factors, including structure formation, biased GC content, and the presence of homopolymers. Single-nucleotide mismatch errors had low biases in HiFi and ONT for all non-B motif types but were increased for G-quadruplexes and Z-DNA in all three technologies. Deletion errors were increased for all non-B types but Z-DNA in Illumina and HiFi, as well as only for G-quadruplexes in ONT. Insertion errors for non-B motifs were highly, moderately, and slightly elevated in Illumina, HiFi, and ONT, respectively. Additionally, we developed a probabilistic approach to determine the number of false positives at non-B motifs depending on sample size and variant frequency, and applied it to publicly available data sets (1000 Genomes, Simons Genome Diversity Project, and gnomAD). We conclude that elevated sequencing errors at non-B DNA motifs should be considered in low-read-depth studies (single-cell, ancient DNA, and pooled-sample population sequencing) and in scoring rare variants. Combining technologies should maximize sequencing accuracy in future studies of non-B DNA.

Molecular simulations of DEAH-box helicases reveal control of domain flexibility by ligands: RNA, ATP, ADP, and G-patch proteins.

DEAH-box helicases use the energy from ATP hydrolysis to translocate along RNA strands. They are composed of tandem RecA-like domains and a C-terminal domain connected by flexible linkers, and the activity of several DEAH-box helicases is regulated by cofactors called G-patch proteins. We used all-atom molecular dynamics simulations of the helicases Prp43, Prp22, and DHX15 in various liganded states to investigate how RNA, ADP, ATP, or G-patch proteins influence their conformational dynamics. The simulations suggest that apo helicases are highly flexible, whereas binding of RNA renders the helicases more rigid. ATP and ADP control the stability of the RecA1-RecA2 interface, but they have only a smaller effect on domain flexibility in absence of a RecA1-RecA2 interface. Binding of a G-patch protein to DHX15 imposes a more structured conformational ensemble, characterized by more defined relative domain arrangements and by an increased conformational stability of the RNA tunnel. However, the effect of the G-patch protein on domain dynamics is far more subtle as compared to the effects of RNA or ATP/ADP. The simulations characterize DEAH-box helicase as dynamic machines whose conformational ensembles are strongly defined by the presence of RNA, ATP, or ADP and only fine-tuned by the presence of G-patch proteins.

Interrogating the Function of Bicistronic Translational Control Elements to Improve Consistency of Gene Expression.

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

Molecular functions of RNA helicases during ribosomal subunit assembly.

During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering their particular functions has long been challenging due to lack of knowledge of their molecular functions and RNA substrates. Advances in the biochemical characterisation ofRNA helicase activities together with new insights into RNA helicase binding sites on pre-ribosomes and structural snapshots of pre-ribosomal complexes containing RNA helicases now open the door to a deeper understanding of precisely how different RNA helicases contribute to ribosomal subunit maturation.

Synergistic Binding of ATP and Nucleic Acids Necessitates UPF1's ATPase Functional Cycle.

UPF1 is a core protein in the nonsense mRNA degradation (NMD) surveillance pathway that degrades aberrant mRNA. UPF1 has both ATPase and RNA helicase activities, but it exhibits mutually exclusive binding of ATP and RNA. This suggests intricate allosteric coupling between ATP and RNA binding that remains unresolved. In this study, we used molecular dynamics simulations and dynamic network analyses to probe the dynamics and free energy landscapes covering UPF1 crystal structures resolved in the Apo state, the ATP bound state, and the ATP-RNA bound (catalytic transition) state. Free energy calculations show that in the presence of ATP and RNA, the transition from the Apo state to the ATP bound state is an uphill process but becomes a downhill process when transitioning to the catalytic transition state. Allostery potential analyses reveal that the Apo and catalytic transition states are mutually allosterically activated toward each other, reflecting the intrinsic ATPase function of UPF1. The Apo state is also allosterically activated toward the ATP bound state. However, binding ATP alone leads to an allosterically trapped state that is difficult to revert to either the Apo or the catalytic transition state. The high allostery potential of Apo UPF1 toward different states results in a "first come, first served" mechanism that requires the synergistic binding of ATP and RNA to drive the ATPase cycle. Our results reconcile UPF1's ATPase and RNA helicase activities within an allostery framework and may apply to other SF1 helicases, as we demonstrate that UPF1's allostery signaling pathways prefer the RecA1 domain over the equally fold-conserved RecA2 domain, and this preference coincides with higher sequence conservation in the RecA1 domain across typical human SF1 helicases.

Identification of Key Genes Related to Immune Cells in Patients with COVID-19 Via Integrated Bioinformatics-Based Analysis.

COVID-19 has spread all over the world which poses a serious threat to social economic development and public health. Despite enormous progress has been made in the prevention and treatment of COVID-19, the specific mechanism and biomarker related to disease severity or prognosis have not been clarified yet. Our study intended to further explore the diagnostic markers of COVID-19 and their relationship with serum immunology by bioinformatics analysis. The datasets about COVID-19 were downloaded from the Gene Expression Omnibus (GEO) dataset. The differentially expressed genes (DEGs) were selected via the limma package. Then, weighted gene co-expression network analysis (WGCNA) was conducted to identify the critical module associated with the clinic status. The intersection DEGs were processed for further enrichment analysis. The final diagnostic genes for COVID-19 were selected and verified through special bioinformatics algorithms. There were significant DEGs between the normal and COVID-19 patients. These genes were mainly enriched in cell cycle, complement and coagulation cascade, extracellular matrix (ECM) receptor interaction, and the P53 signaling pathway. As much as 357 common intersected DEGs were selected in the end. These DEGs were enriched in organelle fission, mitotic cell cycle phase transition, DNA helicase activity, cell cycle, cellular senescence, and P53 signaling pathway. Our study also identified CDC25A, PDCD6, and YWAHE were potential diagnostic markers of COVID-19 with the AUC (area under curve), 0.958 (95% CI 0.920-0.988), 0.941(95% CI 0.892-0.980), and 0.929 (95% CI 0.880-0.971). Moreover, CDC25A, PDCD6, and YWAHE were correlated with plasma cells, macrophages M0, T cells CD4 memory resting, T cells CD8, dendritic cells, and NK cells. Our study discovered that CDC25A, PDCD6, and YWAHE can be used as diagnostic markers for COVID-19. Moreover, these biomarkers were also closely associated with immune cell infiltration, which plays a pivotal role in the diagnosis and progression of COVID-19.

De novo MCM6 variants in neurodevelopmental disorders: a recognizable phenotype related to zinc binding residues

The minichromosome maintenance (MCM) complex acts as a DNA helicase during DNA replication, and thereby regulates cell cycle progression and proliferation. In addition, MCM-complex components localize to centrosomes and play an independent role in ciliogenesis. Pathogenic variants in genes coding for MCM components and other DNA replication factors have been linked to growth and developmental disorders as Meier–Gorlin syndrome and Seckel syndrome. Trio exome/genome sequencing identified the same de novo MCM6 missense variant p.(Cys158Tyr) in two unrelated individuals that presented with overlapping phenotypes consisting of intra-uterine growth retardation, short stature, congenital microcephaly, endocrine features, developmental delay and urogenital anomalies. The identified variant affects a zinc binding cysteine in the MCM6 zinc finger signature. This domain, and specifically cysteine residues, are essential for MCM-complex dimerization and the induction of helicase activity, suggesting a deleterious effect of this variant on DNA replication. Fibroblasts derived from the two affected individuals showed defects both in ciliogenesis and cell proliferation. We additionally traced three unrelated individuals with de novo MCM6 variants in the oligonucleotide binding (OB)-fold domain, presenting with variable (neuro)developmental features including autism spectrum disorder, developmental delay, and epilepsy. Taken together, our findings implicate de novo MCM6 variants in neurodevelopmental disorders. The clinical features and functional defects related to the zinc binding residue resemble those observed in syndromes related to other MCM components and DNA replication factors, while de novo OB-fold domain missense variants may be associated with more variable neurodevelopmental phenotypes. These data encourage consideration of MCM6 variants in the diagnostic arsenal of NDD.

Insights into the Dynamics and Helicase Activity of RecD2 of Deinococcus radiodurans during DNA Repair: A Single-Molecule Perspective.

While the double helix is the most stable conformation of DNA inside cells, its transient unwinding and subsequent partial separation of the two complementary strands yields an intermediate single-stranded DNA (ssDNA). The ssDNA is involved in all major DNA transactions such as replication, transcription, recombination, and repair. The process of DNA unwinding and translocation is shouldered by helicases that transduce the chemical energy derived from nucleotide triphosphate (NTP) hydrolysis to mechanical energy and utilize it to destabilize hydrogen bonds between complementary base pairs. Consequently, a comprehensive understanding of the molecular mechanisms of these enzymes is essential. In the last few decades, a combination of single-molecule techniques (force-based manipulation and visualization) have been employed to study helicase action. These approaches have allowed researchers to study the single helicase-DNA complex in real-time and the free energy landscape of their interaction together with the detection of conformational intermediates and molecular heterogeneity during the course of helicase action. Furthermore, the unique ability of these techniques to resolve helicase motion at nanometer and millisecond spatial and temporal resolutions, respectively, provided surprising insights into their mechanism of action. This perspective outlines the contribution of single-molecule methods in deciphering molecular details of helicase activities. It also exemplifies how each technique was or can be used to study the helicase action of RecD2 in recombination DNA repair.

KREH1 RNA helicase activity promotes utilization of initiator gRNAs across multiple mRNAs in trypanosome RNA editing.

Mitochondrial U-indel RNA editing in kinetoplastid protozoa is directed by trans-acting gRNAs and mediated by a holoenzyme with associated factors. Here, we examine the function of the holoenzyme-associated KREH1 RNA helicase in U-indel editing. We show that KREH1 knockout (KO) impairs editing of a small subset of mRNAs. Overexpression of helicase-dead mutants results in expanded impairment of editing across multiple transcripts, suggesting the existence of enzymes that can compensate for KREH1 in KO cells. In depth analysis of editing defects using quantitative RT-PCR and high-throughput sequencing reveals compromised editing initiation and progression in both KREH1-KO and mutant-expressing cells. In addition, these cells exhibit a distinct defect in the earliest stages of editing in which the initiator gRNA is bypassed, and a small number of editing events takes place just outside this region. Wild type KREH1 and a helicase-dead KREH1 mutant interact similarly with RNA and holoenzyme, and overexpression of both similarly disorders holoenzyme homeostasis. Thus, our data support a model in which KREH1 RNA helicase activity facilitates remodeling of initiator gRNA-mRNA duplexes to permit accurate utilization of initiating gRNAs on multiple transcripts.

Mechanism of 5S RNP recruitment and helicase-surveilled rRNA maturation during pre-60S biogenesis.

Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.

Repurposing of artesunate, an antimalarial drug, as a potential inhibitor of hepatitis E virus.

Hepatitis E virus (HEV) is endemic in several developing countries of Africa and Asia. It mainly causes self-limiting waterborne infections, in either sporadic or outbreak form. Recently, HEV was shown to cause chronic infections in immunosuppressed individuals. Ribavirin and interferon, the current off-label treatment options for hepatitis E, have several side effects. Hence, there is a need for new drugs. We evaluated the antimalarial drug artesunate (ART) against genotype 1 HEV (HEV-1) and HEV-3 using a virus-replicon-based cell culture system. ART exhibited 59% and 43% inhibition of HEV-1 and HEV-3, respectively, at the highest nontoxic concentration. Computational molecular docking analysis showed that ART can bind to the helicase active site (affinity score, -7.4 kcal/mol), indicating its potential to affect ATP hydrolysis activity. An in vitro ATPase activity assay of the helicase indeed showed 24% and 55% inhibition at 19.5 µM (EC50) and 78 µM concentrations of ART, respectively. Since ATP is a substrate of RNA-dependent RNA polymerase (RdRp) as well, we evaluated the effect of ART on the enzymatic activity of the viral polymerase. Interestingly, ART showed 26% and 40% inhibition of the RdRp polymerase activity at 19.5 µM and 78 µM concentrations of ART, respectively. It could be concluded from these findings that ART inhibited replication of both HEV-1 and HEV-3 by directly targeting the activities of the viral enzymes helicase and RdRp. Considering that ART is known to be safe in pregnant women, we think this antimalarial drug deserves further evaluation in animal models.

CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute.

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.

Extended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex

Activating signal co-integrator 1 complex (ASCC) subunit 3 (ASCC3) supports diverse genome maintenance and gene expression processes, and contains tandem Ski2-like NTPase/helicase cassettes crucial for these functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. We present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. TRIP4 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning an ASC-1 homology domain next to the C-terminal helicase cassette of ASCC3, likely supporting substrate engagement and assisting the DNA exit. TRIP4 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC3 for specific processes. Our findings define ASCC3-TRIP4 as a tunable motor module of ASCC that encompasses two cooperating NTPase/helicase units functionally expanded by TRIP4.

Abstract 6179: Comparative study of Polθ helicase and polymerase inhibitor biochemical and cellular potency

Abstract Background: DNA Polymerase θ (Polθ) is active in Theta-mediated end joining (TMEJ), a repair pathway that utilizes resected 3’ ends and micro-homology sequences to repair double strand breaks. Polθ contains an N-terminal helicase domain and a C-terminal polymerase domain. The helicase domain facilitates stripping of RPA/RAD51 from single stranded DNA and is involved in DNA-end recognition, and micro-homology search and pairing. The polymerase domain performs DNA synthesis following micro-homology pairing. Lack of Polθ is synthetic lethal in homologous recombination deficient (HRD) cells. In this study, we utilized existing small molecule inhibitors targeting either helicase or polymerase domain of Polθ, to assess their translational potential in HRD in vitro cancer cell models. Methods: Small molecules inhibiting either the helicase or polymerase activity of Polθ were studied with biochemical and cellular assays. For the biochemical studies, recombinant helicase or polymerase domains were purified. The helicase activity of Polθ was analyzed using ADP-Glo and the polymerase activity by primer extension assay. Binding assays confirmed inhibitor binding to recombinant proteins. To study inhibitor efficacy in cellular models, we probed cell viability of BRCA2-deficient cell lines. Results: Both helicase and polymerase inhibitors showed good potency in inhibiting Polθ enzymatic activity in ATPase or primer extension assays, respectively, with IC50 values in low-nanomolar range. However, the viability of BRCA2-deficient cell lines was not significantly affected after treatment with either helicase or polymerase inhibitors. The measured compound properties (stability, efflux, solubility) did not explain the lack of efficacy in cellular viability assays. Combining Polθ inhibitor treatment with ionizing radiation led to a modest enhancement of radiation-induced decrease in cell viability. Conclusions: Recently, significant advances have been made in Polθ inhibitor development prompted by the synthetic lethal interaction between Polθ and HRD discovered in several screening efforts. Both helicase and polymerase inhibitors are highly potent in biochemical assays. However, without exogenous genotoxic stress, the potential of Polθ inhibitors as therapeutic target may not be reached in vitro. Additional studies are required to understand the translational gap between biochemical and biological read-outs. Citation Format: Johanna K. Ahlskog, Antti Pohjakallio, Ralf Paul, Martin Augustin, Elisabeth Schneider, Sakshi Johri, Tuomas Tervahauta, Hans-Georg Beisel, Lars Neumann, Julia Lindqvist, Anu Moilanen. Comparative study of Polθ helicase and polymerase inhibitor biochemical and cellular potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6179.

Abstract 3098: Structure-based design rules for potent quadruplex-binding compounds based on the naphthalene diimide core

Abstract Quadruplex nucleic acids (G4s) are four-stranded arrangements formed by guanine-rich tracts of DNA or RNA, held together by Hoogsteen hydrogen-bonded guanine quartets. G4 sequences are widely though not randomly distributed in the human genome. They are over-represented in telomeres and in the regulatory regions of many cancer-related genes such as KIT, MYC, VEGF, RAS and HIF. G4 structures can be unwound by helicase activity but can be stabilized by the binding of high-affinity small molecules. The compound QN-302, a tetra-substituted naphthalene diimide (ND) derivative, has nanomolar affinity and selectivity for several DNA and RNA G4s over duplex DNA. QN-302 has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It has significant anti-tumor activity in several models for PDAC - in a MIA-PACA2 xenograft, in the genetic KPC model and in an orthotopic BX-PC3 model. QN-302 is currently in an advanced stage of pre-IND development by Qualigen Inc as a novel anti-cancer agent. QN-302 is the latest in several generations of NDs developed by us and designed by computer modelling based on earlier G4 co-crystal structures determined in this laboratory. The database of seven ND-containing crystal structures includes four different NDs with distinct side chain end groups, all with intramolecular telomeric parallel G4s. End-groups include N-methyl piperazine, hydroxyl, morpholino and N-dimethyl. These structures have been systematically compared for example using least-squares overlays of the structures. All the structures have their four substituents positioned each in a G4 groove, although there are significant differences in orientations of the end groups. Surprisingly, the naphthalene diimide cores do not retain the same orientation in each structure, with three distinct orientations being apparent. Examination of ND core overlap with the adjacent G-quartet has revealed variation in the degree of heterocycle-guanine base overlap, which is dependent on the nature and size of each substituent. However, all structures show minimal ND-guanine overlap, with typically just one out of four guanines in a G-quartet being involved. This strongly implies that most of the contribution to ND-G4 affinity comes from the side chains, and especially from the protonated end groups. These and other observations have led us to develop a structure-activity framework for tetra-substituted NDs, which has culminated in their optimization and the design of QN-302, with enhanced cellular potency and G4 affinity compared to previous NDs. Citation Format: Stephen Neidle. Structure-based design rules for potent quadruplex-binding compounds based on the naphthalene diimide core [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3098.

Abstract 4479: Targeting of eIF4A1 curtails lung metastases in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer. Patients diagnosed with TNBC benefit less or none from any current targeted therapies and also the standard-of-care neoadjuvant chemotherapies (NACT) which is cytotoxic. TNBC shows an increased risk of tumor relapse, therapy failure, and high proclivity to develop distant metastases. Intrinsic resistance to NACT is mainly due to a small population of breast cancer cells that acquire stem cell-like features (BCSCs). BCSCs are capable of self-renewal, proliferation, plasticity, immune regulation and chemoresistance. These properties mediate tumorigenesis, metastasis, and resistance to NACT and sometimes even to targeted therapy. Overall, FDA-approved drugs that are effective for advanced TNBC are currently unavailable. The eukaryotic translation initiation factor (eIF4A1) is an mRNA helicase that catalyzes the ATP-dependent unwinding of many oncogenic mRNAs which harbor secondary structures in the 5’ leader region. There is preferential reliance on eIF4A1 by tumor cells as many oncogenic mRNAs require the helicase activity of eIF4A1 for their efficient translation. Several eIF4A1-dependent mRNAs (downstream effectors) include: survivin (survival), c-MYC, STAT1 (immunoevasion through transcription and subsequent translation of PD-L1), Rho kinase1 (invasion), and Cyclins D1 and D3. Bioinformatic analyses revealed that higher gene expression of eIF4A1 significantly impacts patient survival. Clinically, an enhanced protein expression of eIF4A1 was evident in histopathologic analyses of drug-resistant human TNBC biospecimens. Importantly, our in vitro data indicated that the viability of BCSCs was significantly reduced when eIF4A1 is targeted by nanomolar levels of Rocaglamide A (RocA). Furthermore, in paclitaxel-resistant TNBC cells, there is an upregulation in the total levels of eIF4A1 and its downstream effectors. Pluripotency transcription factors (e.g., SOX2, OCT4 and NANOG) and drug transporters (ABCB1, ABCG2 and ABCC1) were also increased in an eIF4A1-dependent manner. Genetic ablation and pharmacological targeting of eIF4A1 with RocA reversed the drug-resistant profile. Our in vitro data implied a key role for eIF4A1 in chemoresistance/therapy failure. Targeting of eIF4A1 may be an effective anti-cancer strategy to overcome drug resistance. To this end, we orthotopically implanted CRISPR-control (CC) and eIF4A1-KO (KO) human TNBC cells and evaluated the primary tumor progression and metastases in NOD/SCID-beige mice. Longitudinal tumor progression was followed by IVIS imaging. The tumor volume and the wet weight was significantly higher in CC than KO tumors (n=7 mice). Similarly, the lung metastatic burden is significantly low in KO than CC mice (n=7 mice; p<0.002)). Taken together, we propose that eIF4A1 is an actionable molecular target for drug-resistant TNBC using chemotherapy/immunotherapy. Citation Format: Sangita Sridharan, Shobhit Srivastava, David Terrero, Saloni Malla, Amit K. Tiwari, Dayanidhi Raman. Targeting of eIF4A1 curtails lung metastases in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4479.

Helicase activities of Rad5 and Rrm3 genetically interact in the prevention of recombinogenic DNA lesions in Saccharomyces cerevisiae

The genome must be monitored to ensure its duplication is completed accurately to prevent genome instability. In Saccharomyces cerevisiae, the 5′ to 3′ DNA helicase Rrm3, a member of the conserved PIF1 family, facilitates replication fork progression through an unknown mechanism. Disruption of Rrm3 helicase activity leads to increased replication fork pausing throughout the yeast genome. Here, we show that Rrm3 contributes to replication stress tolerance in the absence of the fork reversal activity of Rad5, defined by its HIRAN domain and DNA helicase activity, but not in the absence of Rad5′s ubiquitin ligase activity. The Rrm3 and Rad5 helicase activities also interact in the prevention of recombinogenic DNA lesions, and DNA lesions that accumulate in their absence need to be salvaged by a Rad59-dependent recombination pathway. Disruption of the structure-specific endonuclease Mus81 leads to accumulation of recombinogenic DNA lesions and chromosomal rearrangements in the absence of Rrm3, but not Rad5. Thus, at least two mechanisms exist to overcome fork stalling at replication barriers, defined by Rad5-mediated fork reversal and Mus81-mediated cleavage, and contribute to the maintenance of chromosome stability in the absence of Rrm3.