PIM kinase inhibitors: an updated patent review (2016-present).

INTRODUCTION: PIM Kinases (PIM-1, PIM-2, and PIM-3) have been reported to play crucial role in signaling cascades that govern cell survival, proliferation, and differentiation. Over-expression of these kinases leads to hematological malignancies such as diffuse large B cell lymphomas (DLBCL), multiple myeloma, leukemia, lymphoma and prostate cancer etc. PIM kinases as biomarkers and potential therapeutic targets have shown promise toward precision cancer therapy. The selective PIM-1, PIM-2, and/or PIM-3 isoform inhibitors have shown significant results in patients with advanced stages of cancer including relapsed/refractory cancer. AREAS COVERED: A comprehensive literature review of PIM Kinases (PIM-1, PIM-2, and PIM-3) in oncogenesis, the patented PIM kinase inhibitors (2016-Present), and their pharmacological and structural insights have been highlighted. EXPERT OPINION: Recently, PIM kinases viz. PIM-1, PIM-2, and PIM-3 (members of the serine/threonine protein kinase family) as therapeutic targets have attracted considerable interest in oncology especially in hematological malignancies. The patented PIM kinase inhibitors comprised of heterocyclic (fused)ring structure(s) like indole, pyridine, pyrazine, pyrazole, pyridazine, piperazine, thiazole, oxadiazole, quinoline, triazolo-pyridine, pyrazolo-pyridine, imidazo-pyridazine, oxadiazole-thione, pyrazolo-pyrimidine, triazolo-pyridazine, imidazo-pyridazine, pyrazolo-quinazoline and pyrazolo-pyridine etc. showed promising results in cancer chemotherapy.

Recent Advances in the Treatment and Management of Alzheimer's Disease: A Precision Medicine Perspective.

About 60% to 70% of people with dementia have Alzheimer's Disease (AD), a neuro-degenerative illness. One reason for this disorder is the misfolding of naturally occurring proteins in the human brain, specifically ß-amyloid (Aß) and tau. Certain diagnostic imaging techniques, such as amyloid PET imaging, tau PET imaging, Magnetic Resonance Imaging (MRI), Comput-erized Tomography (CT), and others, can detect biomarkers in blood, plasma, and cerebral spinal fluids, like an increased level of ß-amyloid, plaques, and tangles. In order to create new pharma-cotherapeutics for Alzheimer's disease, researchers must have a thorough and detailed knowledge of amyloid beta misfolding and other related aspects. Dolopezil, rivastigmine, galantamine, and other acetylcholinesterase inhibitors are among the medications now used to treat Alzheimer's disease. Another medication that can temporarily alleviate dementia symptoms is memantine, which blocks the N-methyl-D-aspartate (NMDA) receptor. However, it is not able to halt or re-verse the progression of the disease. Medication now on the market can only halt its advance-ment, not reverse it. Interventions to alleviate behavioral and psychological symptoms, exhibit an-ti-neuroinflammation and anti-tau effects, induce neurotransmitter alteration and cognitive en-hancement, and provide other targets have recently been developed. For some Alzheimer's pa-tients, the FDA-approved monoclonal antibody, aducanumab, is an option; for others, phase 3 clinical studies are underway for drugs, like lecanemab and donanemab, which have demonstrat-ed potential in eliminating amyloid protein. However, additional study is required to identify and address these limitations in order to reduce the likelihood of side effects and maximize the thera-peutic efficacy.

Recent Advances of PI3 Kinase Inhibitors: Structure Anticancer Activity Relationship Studies.

Phosphatidyl-inositol-3-kinase (PI3K) has emerged as a potential therapeutic target for the development of novel anticancer drugs. The dysregulation of PI3K has been associated with many human malignancies such as breast, colon, endometrial, brain, and prostate cancers. The PI3K kinases in their different isoforms namely α, ß, δ, and γ, encode PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes. Specific gene mutation or overexpression of the protein is responsible for therapeutic failure of current therapeutics. Recently, various PI3K signaling pathway inhibitors have been identified which showed promising therapeutic results by acting on specific isoforms of the kinase too. Several inhibitors containing medicinally privileged scaffolds like oxadiazole, pyrrolotriazine, quinazoline, quinazolinone, quinazoline-chalcone hybrids, quinazoline-sulfonamide, pyrazolochalcone, quinolone hydroxamic acid, benzofuropyridinone, imidazopyridine, benzoxazines, dibenzoxanthene, indoloderivatives, benzimidazole, and benzothiazine derivatives have been developed to target PI3K pathway and/or a specific isoform. The PI3K inhibitors which are under clinical trial studies include GDC-0032, INK1117 for PI3K-α, and AZD8186 for PI3K-ß. This review primarily focuses on the structural insights and structure anticancer activity relationship studies of recent PI3K inhibitors including their clinical stages of development and therapeutic values.

Recent Advances in PI3 Kinase Inhibitors: Anticancer Activities and Structure-Activity Relationships.

Phosphatidyl-inositol-3-kinase (PI3K) has emerged as a potential therapeutic target for the development of novel anticancer drugs. The dysregulation of PI3K has been associated with many human malignancies such as breast, colon, endometrial, brain, and prostate cancers. The PI3K kinases in their different isoforms, namely α, ß, δ, and γ, encode PIK3CA, PIK3CB, PIK3CD, and PIK3CG genes. Specific gene mutation or overexpression of the protein is responsible for the therapeutic failure of current therapeutics. Recently, various PI3K signaling pathway inhibitors have been identified, which showed promising therapeutic results by acting on specific isoforms of the kinase too. Several inhibitors containing medicinally privileged scaffolds like oxadiazole, pyrrolotriazine, quinazoline, quinazolinone, quinazoline-chalcone hybrids, quinazoline-sulfonamide, pyrazolochalcone, quinolone hydroxamic acid, benzofuropyridinone, imidazopyridine, benzoxazines, dibenzoxanthene, indoloderivatives, benzimidazole, and benzothiazine derivatives have been developed to target the PI3K pathway and/or a specific isoform. The PI3K inhibitors under clinical trial studies include GDC-0032, INK1117 for PI3K-α, and AZD8186 for PI3K-ß. This review primarily focuses on the structural insights, anticancer activities, and structure-activity relationship (SARs) studies of recent PI3K inhibitors, including their clinical stages of development and therapeutic values.

Materialization of novel composite bio-based active edible film functionalized with essential oils on antimicrobial and antioxidative aspect of chicken nuggets during extended storage.

The present study was aimed to develop Manihot esculenta and Carrageenan bio-based composite active film functionalized with anise, caraway, and nutmeg essential oils (EOs) and to assess the shelf life of chicken nuggets wrapped with the developed film at refrigeration storage. Overall, the Minimum Inhibitory Concentration (MIC) values of the three EOs ranged from 0.4 to 0.8% v/v of which nutmeg EO was found most effective. Incorporation of EOs in the film resulted in significant (P ˂ 0.05) decrease in tensile strength and water activity while elongation at break was significantly (P ˂ 0.05) increased. No significant (P ˃ 0.05) changes in thickness and Water Vapor Transmission Rate (WVTR) were observed. Based on physicomechanical and sensory evaluation, films incorporated with 0.5, 1, and 1% concentration of anise, nutmeg, and caraway EO were selected. Chicken nuggets overwrapped with aforementioned films were stored aerobically at refrigeration temperature (4 ± 1 °C) to evaluate antimicrobial, antioxidant, and sensory characteristics. The result indicated that pH, peroxide, free fatty acid (FFA), and thiobarbituric acide (TBA) value of treatments were significantly (P ˂ 0.05) lower than controls however significantly (P < 0.05) higher DPPH activity was observed in all treatments. The total plate count, psychrophilic count and, yeast and mold count were also significantly (P ˂ 0.01) lower in treatment groups and were within the permissible limits. The treated samples were well acceptable during whole storage period of 15 days. The application of composite, active edible bio-based film was found proficient in confining product quality attributes throughout storage. PRACTICAL APPLICATION: The majority of films used for packaging of meat and meat products are derived from synthetic "plastic" materials. The demerits associated with plastics have eventually led to explore natural alternatives such as edible films. The composite-active bio-based films have a huge potential to be molded for specific film properties based on requirements of product-specific packaging conditions.

Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro.

As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.

PIM kinase inhibitors: Structural and pharmacological perspectives.

The PIM kinase, also known as serine/threonine kinase plays an important role in cancer biology and is found in three different isoforms namely PIM-1, PIM-2, and PIM-3. They are extensively distributed and are implicated in a variety of biological processes, including cell proliferation, cell differentiation, and apoptosis. They act as weak oncogene and whenever expressed in exacerbating forms are responsible for different types of human cancer. Recently, different isoforms of PIM kinase have been identified as a clinical biomarker and potential therapeutic target for personalized treatment of advanced cancer. The inhibition of PIM kinase has become a scientific interest and some inhibitors have been developed and/or are under different phases of clinical trials. Several medicinally privileged heterocyclic ring scaffolds such as pyrrole, pyrimidine, thiazolidine, benzofuran, indole, triazole, oxadiazole, and quinoline derivatives have been synthesized and evaluated for their PIM inhibitory activity. This review comprehensively focuses on pharmacological implications of PIM kinases in oncogenesis, structural insights of PIM inhibitors and their structure-activity relationships (SARs).

An updated patent review of therapeutic applications of chalcone derivatives (2014-present).

INTRODUCTION: Chalcone or benzylideneacetophenone or 1,3-diphenyl-2-propene-1-one is a natural product comprising of two aromatic rings connected together by a three-carbon α, ß unsaturated carbonyl bridge. It is one of the most privileged scaffolds in medicinal chemistry that can be synthesized in the laboratory and can be converted into several therapeutically active heterocyclic scaffolds. It exhibits multifarious pharmacological activities and also plays a key role in several non-pharmacological scientific applications. AREAS COVERED: The present article comprehensively focuses on the approved patents during the time duration 2014-2018 on various chalcone molecules with diverse pharmacological activities. EXPERT OPINION: The study puts forward the latest updated therapeutic applications of chalcone-based compounds as antiproliferative, antidiabetic, anti-infective, anti-inflammatory, antioxidant, antiaging, neuroprotective, and cardioprotective agents. The type, position, and the number of substituents present on the chalcone scaffold have been perceived to play an imperative function in interacting with molecular targets (receptor, enzyme, and/or channel) to express the biological responses. In the majority of the studies, the overall activity of the ligand administered as pharmaceutically acceptable salt is found to be better than that of standard marketed drug preparation. The article will certainly attract (medicinal)-chemists actively involved in the development of therapeutically active scaffolds.

Perspectives of medicinally privileged chalcone based metal coordination compounds for biomedical applications.

Recent clinical reports have highlighted the increasing occurrence of drug resistance of known therapeutics. Particularly, antibiotic resistant microorganisms and multidrug resistance have posed a serious threat to health of the people. Since ages, metals and metal complexes have played key role in the development of contemporary chemotherapy. Many organic compounds used in medicine do not have a purely organic mode of action and require traces of metal ions directly or indirectly for activation or biotransformation. For decades, the metallopharmaceuticals have attracted researchers across the globe due to their amplified therapeutic/modulatory effect by altering the pharmacokinetic and pharmacodynamic properties of the complexes towards biological receptors. Medicinally privileged natural and (semi)-synthetic chalcones have already been reported to possess a wide variety of pharmacological effects by modulating diverse molecular targets. The presence of carbonyl, hydroxyl, phenolic oxygen and/or heteroatom(s) in heterocyclic ring system makes them excellent chelating ligand for metal coordination. Particularly, the metal complexes of bidentate chalcone/Schiff base analogs and ferrocenyl chalcones have shown great potential. In this review, the chelating/coordinating property of substituted chalcones, the therapeutic, catalytic, chemosensing and photosensitizing potential of various metal-chalcone complexes, their structural features and structure activity relationships (SARs) have been highlighted. Further, the understanding of coordination mode, their stoichiometric characteristics, and mode of action(s), this review may be helpful for medicinal and bioinorganic chemists to design and develop novel, more potent, safe, selective and cost-effective chalcone-based coordination compounds for diverse biomedical applications.

Warsaw breakage syndrome: Further clinical and genetic delineation.

Warsaw breakage syndrome (WBS) is a recently recognized DDX11-related rare cohesinopathy, characterized by severe prenatal and postnatal growth restriction, microcephaly, developmental delay, cochlear anomalies, and sensorineural hearing loss. Only seven cases have been reported in the English literature, and thus the information on the phenotype and genotype of this interesting condition is limited. We provide clinical and molecular information on five additional unrelated patients carrying novel bi-allelic variants in the DDX11 gene, identified via whole exome sequencing. One of the variants was found to be a novel Saudi founder variant. All identified variants were classified as pathogenic or likely pathogenic except for one that was initially classified as a variant of unknown significance (VOUS) (p.Arg378Pro). Functional characterization of this VOUS using heterologous expression of wild type and mutant DDX11 revealed a marked effect on protein stability, thus confirming pathogenicity of this variant. The phenotypic data of the seven WBS reported patients were compared to our patients for further phenotypic delineation. Although all the reported patients had cochlear hypoplasia, one patient also had posterior labyrinthine anomaly. We conclude that while the cardinal clinical features in WBS (microcephaly, growth retardation, and cochlear anomalies) are almost universally present, the breakage phenotype is highly variable and can be absent in some cases. This report further expands the knowledge of the phenotypic and molecular features of WBS.

A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair.

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

Development of sweet and sour chicken meat spread based on sensory attributes: process optimization using response surface methodology.

Now a day's meat and meat products are not only generating convenience trends; they have been recognized as core of meat industry. Meat spread is a convenience cooked spreadable product prepared with meat and non-meat additives. Response surface methodology was used to investigate the effects of three different levels of honey (10, 15, 20 g), vinegar (2.0, 6.0, 10.0 ml) and tomato powder (0.5, 1.0, 1.5 g) on response variables viz. color/appearance, flavor, spreadability, texture, after taste, adhesiveability, overall acceptability, while standardizing the process of development of sweet and sour chicken meat spread box-behnken experimental design was used in which 17 different runs with 5 trials of three similar centre point. A second order polynomial was fitted to all the response variables and surface plots as well as equations were conducted. All the processing variables significantly affected the response variables either linearly or quadratically whereas the "Lack of Fit" was non-significant relative to the pure error. For optimization, target values were set in the form of ranges of all the processing and response variables. While applying multiple regression analysis, a total of 43 workable solutions was found, out of which the product with 14.28% honey, 5.38% vinegar and 1.39% tomato powder was selected. The responses for color/appearance, flavor, spreadability, texture, after taste, adhesiveability, overall acceptability, were predicted at 7.11, 6.72, 7.00, 6.99, 6.61, 6.94 and 6.79 respectively, with a desirability value of 1.

Chalcone Derivatives: Anti-inflammatory Potential and Molecular Targets Perspectives.

Chalcone or (E)-1,3-diphenyl-2-propene-1-one scaffold has gained considerable scientific interest in medicinal chemistry owing to its simple chemistry, ease in synthesizing a variety of derivatives and exhibiting a broad range of promising pharmacological activities by modulating several molecular targets. A number of natural and (semi-) synthetic chalcone derivatives have demonstrated admirable anti-inflammatory activity due to their inhibitory potential against various therapeutic targets like Cyclooxygenase (COX), Lipooxygenase (LOX), Interleukins (IL), Prostaglandins (PGs), Nitric Oxide Synthase (NOS), Leukotriene D4 (LTD4), Nuclear Factor-κB (NF- κB), Intracellular Cell Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), Monocyte Chemoattractant Protein-1 (MCP-1) and TLR4/MD-2, etc. The chalcone scaffold with hydroxyl, methoxyl, carboxyl, prenyl group and/or heterocyclic ring substitution like thiophene/furan/indole showed promising anti-inflammatory activity. In this review, a comprehensive study (from the year 1991 to 2016) on multi-targets of inflammatory interest, related inflammation reactions and their treatment by chalcone-based inhibitors acting on various molecular targets entailed in inflammation, Structure-Activity Relationships (SARs), Mechanism of Actions (MOAs), and patents are highlighted.

MEK inhibitors in oncology: a patent review (2015-Present).

INTRODUCTION: The RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways have been identified as promising therapeutic targets for cancer therapy. Over-activation of these pathways and their components including gene mutations has been considered as one of the major causes of melanoma. Mitogen-activated protein kinase (MEK) is a downstream kinase of RAS pathway found in two different forms MEK1/2. The MEK inhibitors in combination with other kinase/mutant gene inhibitors have shown promising results in patients with metastatic melanoma. Areas covered: A comprehensive review of the patent literature (2015 - Present) on MEK inhibitors, their combinations with other kinase inhibitors and structural insights has been highlighted. Expert opinion: Recently mitogen-activated protein kinase (MEK) inhibitors have attracted considerable interest in oncology especially in melanoma. The MEK inhibitors showed promising results in patients with metastatic melanoma harboring mutant genes such as BRAF, KRAS. The MEK1/2 inhibitors in combination with BRAF, KRAS and/or PI3K inhibitors showed promising results in mutated colorectal, pancreatic adenocarcinoma, solid tumor, and relapsed/refractory melanoma. The combination delays the onset of acquired resistance, resulting in increased progression-free and overall survival. The combination and/or multi-targeted kinase/mutant gene inhibitors may be a therapeutic option for the personalized cancer treatment of patients with relapsed or refractory multiple myeloma.

K-Ras and its inhibitors towards personalized cancer treatment: Pharmacological and structural perspectives.

The discovery of genetic, genomic and clinical biomarkers have revolutionized the treatment option in the form of personalized medicine which allows to accurately predict a person's susceptibility/progression of disease, the patient's response to therapy, and maximize the therapeutic outcome in terms of low/no toxicity for a particular patient. Recently, the U.S. Food and Drug Administration has realized the contribution of pharmacogenomics in better healthcare and advocated the consideration of pharmacogenomic principles in making safer and more effective drug. Many anticancer drugs show reduced or no response in cancer patients with tumor specific gene mutations such as B-Raf and K-Ras. The high incidence of K-Ras mutation has been reported in pancreatic, colon, and lung carcinomas. The identification of K-Ras as a clinical biomarker and potential therapeutic target has attracted the scientific community to develop effective and precise anticancer drug. Inhibitors which block farnesylation of Ras have been developed or under clinical trial studies. Tipifarnib, approved by USFDA for the treatment of elderly acute leukemia is a Ras pathway inhibitor. Some peptidomimetics and bi-substrate inhibitors like FTI 276, FTI 277, B956, B1086, L731, L735, L739, L750, BMS-214662, L778123, and L778123 are under clinical trials. Recently mutant K-Ras has been considered as potential biomarker and target for precise cancer therapy. This review focuses primarily on the Ras/Raf/MEK/ERK signaling pathway including K-Ras mutation as therapeutic target, inhibitors and their structure activity relationships (SARs) for the design and development of anticancer agents.

Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA.

Cockayne syndrome is a neurodegenerative accelerated aging disorder caused by mutations in the CSA or CSB genes. Although the pathogenesis of Cockayne syndrome has remained elusive, recent work implicates mitochondrial dysfunction in the disease progression. Here, we present evidence that loss of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number of cell lines. Furthermore, machine-learning algorithms predict that diseases with defects in ribosomal DNA (rDNA) transcription have mitochondrial dysfunction, and, accordingly, this is found when factors involved in rDNA transcription are knocked down. Mechanistically, loss of CSA or CSB leads to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans In conclusion, this work supports a role for impaired ribosomal DNA transcription in Cockayne syndrome and suggests that transcription-coupled resolution of secondary structures may be a mechanism to repress spurious activation of a DNA damage response.

A Long Noncoding RNA Regulates Sister Chromatid Cohesion.

Long noncoding RNAs (lncRNAs) are involved in diverse cellular processes through multiple mechanisms. Here, we describe a previously uncharacterized human lncRNA, CONCR (cohesion regulator noncoding RNA), that is transcriptionally activated by MYC and is upregulated in multiple cancer types. The expression of CONCR is cell cycle regulated, and it is required for cell-cycle progression and DNA replication. Moreover, cells depleted of CONCR show severe defects in sister chromatid cohesion, suggesting an essential role for CONCR in cohesion establishment during cell division. CONCR interacts with and regulates the activity of DDX11, a DNA-dependent ATPase and helicase involved in DNA replication and sister chromatid cohesion. These findings unveil a direct role for an lncRNA in the establishment of sister chromatid cohesion by modulating DDX11 enzymatic activity.

Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles.

Mounting evidence indicates that alternate DNA structures, which deviate from normal double helical DNA, form in vivo and influence cellular processes such as replication and transcription. However, our understanding of how the cellular machinery deals with unusual DNA structures such as G-quadruplexes (G4), triplexes, or hairpins is only beginning to emerge. New advances in the field implicate a direct role of the Fanconi Anemia Group J (FANCJ) helicase, which is linked to a hereditary chromosomal instability disorder and important for cancer suppression, in replication past unusual DNA obstacles. This work sets the stage for significant progress in dissecting the molecular mechanisms whereby replication perturbation by abnormal DNA structures leads to genomic instability. In this review, we focus on FANCJ and its role to enable efficient DNA replication when the fork encounters vastly abundant naturally occurring DNA obstacles, which may have implications for targeting rapidly dividing cancer cells.

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING.

Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.