Inhibition Of Polymerase Activity Research Articles

Exploring Bacterial Diversity: How Far Have We Reached?

Abstract Many methods have been developed for studying and comparing bacterial diversity. These methods suffer from a number of drawbacks. Culture-dependent methods have a drawback that only a small number of bacteria can be cultured. Although many modifications in the traditional cultivation approach have been made, such as the use of gellan instead of agar and high throughput dilution to extinction culturing, but a large fraction of microbes still remain uncultured. Culture-independent methods were developed to explore uncultured bacterial diversity but they have their own drawbacks. PCR-based methods have biases during DNA extraction and the removal of substances that may inhibit polymerase activity during PCR and digestion with restriction enzymes. “Omics” approach, i.e., metagenomics, metatranscriptomics, and metaproteomics, aim to link bacterial community structure with function. Different combinations of methods can be used to know more precisely about the bacterial diversity. To date, no known method can reveal the exact bacterial diversity of different environments. This lacuna needs to be filled and newer methods must be developed that can help in exploring the immense bacterial diversity created by nature.

Stabilizing Pseudouridimycin: Synthesis, RNA Polymerase Inhibitory Activity, and Antibacterial Activity of Dipeptide-Modified Analogues.

Pseudouridimycin (PUM) is a microbially produced C-nucleoside dipeptide that selectively targets the nucleotide addition site of bacterial RNA polymerase (RNAP) and that has a lower rate of spontaneous resistance emergence relative to current drugs that target RNAP. Despite its promising biological profile, PUM undergoes relatively rapid decomposition in buffered aqueous solutions. Here, we describe the synthesis, RNAP-inhibitory activity, and antibacterial activity of chemically stabilized analogues of PUM. These analogues feature targeted modifications that mitigate guanidine-mediated hydroxamate bond scission. A subset of analogues in which the central hydroxamate is replaced with amide or hydrazide isosteres retain the antibacterial activity of the natural product.

Polyamines and eIF5A hypusination facilitate SREBP2 synthesis and cholesterol production leading to enhanced enterovirus attachment and infection.

Metabolism is key to cellular processes that underlie the ability of a virus to productively infect. Polyamines are small metabolites vital for many host cell processes including proliferation, transcription, and translation. Polyamine depletion also inhibits virus infection via diverse mechanisms, including inhibiting polymerase activity and viral translation. We showed that Coxsackievirus B3 (CVB3) attachment requires polyamines; however, the mechanism was unknown. Here, we report polyamines' involvement in translation, through a process called hypusination, promotes expression of cholesterol synthesis genes by supporting SREBP2 synthesis, the master transcriptional regulator of cholesterol synthesis genes. Measuring bulk transcription, we find polyamines support expression of cholesterol synthesis genes, regulated by SREBP2. Thus, polyamine depletion inhibits CVB3 by depleting cellular cholesterol. Exogenous cholesterol rescues CVB3 attachment, and mutant CVB3 resistant to polyamine depletion exhibits resistance to cholesterol perturbation. This study provides a novel link between polyamine and cholesterol homeostasis, a mechanism through which polyamines impact CVB3 infection.

Phase II Trials of Iniparib (BSI-201) in Combination with Gemcitabine and Carboplatin in Patients with Recurrent Ovarian Cancer.

Iniparib (BSI-201), a novel anticancer agent thought to have poly(ADP-ribose) polymerase (PARP) inhibitory activity and synergy with both gemcitabine and carboplatin (GC) was evaluated in 2 cohorts with GC. Parallel multicenter, single-arm, phase II studies using a Simon two-stage design. Eligible patients had a histological diagnosis of epithelial ovarian carcinoma, fallopian tube cancer, or primary peritoneal carcinoma and demonstration of platinum-sensitive (≥6 months [mo]) or -resistant disease (relapse 2-6 mo post-platinum). Carboplatin (AUC 4 IV day 1), gemcitabine (1000 mg/m2 IV days 1 and 8), and iniparib (5.6 mg/kg IV days 1, 4, 8, and 11) were given on a 21-day cycle. The overall response rate (ORR RECIST 1.0) in platinum sensitive disease was 66% (95% CI, 49-80) with a higher response rate in the 15 pts with germline BRCA mutations (gBRCAmut) (73%). Median PFS was 9.9 (95% CI, 8.2-11.3) months. In the platinum resistant population the ORR was 26% (95% CI, 14-42), however in the 11 pts for whom BRCA mutation was present, the best overall response was PR in 5 (46%). Median PFS was 6.8 months (range, 5.7-7.7 months). Notably, among the 17 CA-125-response-evaluable patients who did not achieve tumor response, 7 (41.2%) patients had a CA125 response, and 93% has clinical benefit (CR + PR + SD). The GCI combination was generally well tolerated despite a high incidence of thrombocytopenia and neutropenia, with no new toxicities. Given the subsequent lack of efficacy demonstrated for iniparib in breast cancer, these are studies of GC and demonstrate a higher than traditionally appreciated activity in patients with platinum-sensitive and -resistant recurrent ovarian cancer, especially in patients that harbor a BRCA mutation, resetting the benchmark for efficacy in phase II trials. (ClinicalTrials.gov Identifiers: NCT01033292 & NCT01033123).

Improved PCR by the Use of Disruptors, a New Class of Oligonucleotide Reagents.

As a powerful tool, polymerase chain reaction (PCR) has been indispensable and widely used in a large array of applications. In practice, many factors may affect the overall performance of a PCR. One such factor is the stability of intramolecular secondary structure formed within single-stranded template. The higher the stability of such a structure, the more likely it will have adverse effects on PCR performance. Traditionally, chemical reagents believed to reduce the stability of nucleic acid secondary structures, such as DMSO and betaine, have been used to mitigate their adverse effects on PCR performance. However, these reagents have apparent downsides including increasing replication error rate, inhibiting polymerase activity, and being ineffective against secondary structures of very high stabilities. Disruptors, a new class of oligonucleotide reagents, do not exhibit such downsides. They are specifically designed to target intramolecular secondary structures only without any effect on the replication of other regions of the template. Their effective concentration range for improving PCR performance is well tolerated by PCR. And they are very effective in improving PCR performance on templates that are notoriously difficult to amplify by PCR even in the presence of DMSO or betaine, e.g., the inverted terminal repeat of adeno-associated virus (AAV-ITR). In this chapter, the application of disruptors in PCR is described with AAV-ITR as the example template.

New Route to the Synthesis of Novel Pyrazolo[1,5-a]pyrimidines and Evaluation of their Antimicrobial Activity as RNA Polymerase Inhibitors.

The current study aimed to synthesize novel pyrazolo[1,5-a]pyrimidines based on 5- aminopyrazoles 3, evaluate their antimicrobial activity, and study the minimum inhibitory concentration (MIC) for the most active compounds. In addition, molecular docking studies and RNA polymerase inhibitory activity were determined. Starting with our previously reported 5-aminopyrazoles 3, a number of novel pyrazolo[1,5- a]pyrimidines were synthesized. Due to the similarity of pyrazolopyrimidine derivatives with the purine systems, pyrazolopyrimidines are important in many different biological applications, most notably as anti-tumor, antibacterial, and hepatitis C virus inhibitors. The pharmaceutical applications of the pyrazolopyrimidine derivatives were explained in several approved drugs like Indiplon, Zaloplan, and Ocinaplon. To prepare a novel antimicrobial agent, namely pyrazolo[1,5-a]pyrimidine, reveal their structures using different spectral data, the minimum inhibitory concentration (MIC) for the most active compounds was evaluated, and both the molecular docking and the RNA polymerase inhibitory activity were determined. A number of different pyrazolopyrimidines namely 2-(phenylamino)-6,11-dihydrobenzo[g]pyrazolo [1,5-a]quinazoline-3-carboxamides (5a-c), (E)-5,7-dimethyl-2-(phenylamino)-6-(phenyldiazenyl)pyrazolo-[1,5- a]pyrimidine-3-carboxamides (7a-c), 7-amino-2-(phenylamino) pyrazolo[1,5-a]pyrimidine-3-carboxamides (11af), 7-amino-2-(phenylamino)-5-(2-thienyl)pyrazolo[1,5-a]pyrimidine-3-carboxamides (14-f) and ethyl 7-amino-3- carbamoyl-2-(phenylamino)-5-(4-pyridyl)pyrazolo[1,5-a]pyrimidine-6-carboxylate derivatives (14g-i) were synthesized through the reaction of 5-aminopyrazoles 3 with a variety of chemical reagents. On the other hand, the evaluation of the antimicrobial activity for all the prepared compounds was screened through different strains as Gram-positive bacteria, such as staphylococcus aureus and Streptococcus mutans, and Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and klebsiella. The antifungal activity was determined by Candids Albicans fungal strain, and the MIC of the most active compounds was measured. The molecular docking was recorded, and the RNA polymerase inhibitory activity was estimated for the high docking score compounds. Compounds 5a, 5b, 5c, 7a, 7b, 7c, 11d, 14b, and 14h were the most active compounds against some of the bacterial and fungal tested strains. MIC was determined for the most active tested compounds. As an antimicrobial agent, compound 7b was the most potent, with a high docking score and RNA polymerase inhibitory activity (IC50= 0.213 μg/ml) compared to Rifampicin (IC50= 0.244 μg/ml). The reactivity of the latter compound was attributed to the presence of 4-Br-C6H4 moiety. The results demonstrated that docking studies on the most active compounds in the RNA polymerase active site were consistent with in vitro assays. The resultant novel bioactive pyrazolo[1,5-a]pyrimidine derivatives were synthesized based on 5- aminopyrazole derivatives 3. The current study evaluated the antimicrobial activity for all the prepared compounds, followed by the determination of the MIC for the most potent active compounds. The molecular docking study was performed, and it was appropriate with the in vitro activity. The RNA polymerase inhibitory activity was assessed for the most active antimicrobial compounds with a high docking score (7b, 7c, 14a, 14b, 14e, 14i). Compound 7b was the most potent compound inhibiting RNA polymerase enzyme compared to the reference drug Rifampicin. Other: The novel prepared heterocyclic systems are extremely important in a variety of domains, especially biological and pharmacological ones.

Nucleoporin 85 interacts with influenza A virus PB1 and PB2 to promote its replication by facilitating nuclear import of ribonucleoprotein.

Transcription and replication of the influenza A virus (IAV) genome take place in the nucleus of infected cells, which rely on host factors to aid viral ribonucleoprotein (vRNP) to cross the nuclear pore complex (NPC) and complete the bidirectional nucleocytoplasmic trafficking. Here, we showed that nucleoporin 85 (NUP85), a component of NPC, interacted with RNP subunits polymerase basic 1 (PB1) and polymerase basic 2 (PB2) in an RNA-dependent manner during IAV infection. Knockdown of NUP85 delayed the nuclear import of vRNP, PB1 and PB2, inhibiting polymerase activity and ultimately suppressing viral replication. Further analysis revealed that NUP85 assisted the binding of PB1 to nuclear transport factor Ran-binding protein 5 (RanBP5) and the binding of PB2 to nuclear transport factor importin α1 and importin α7. We also found that NUP85 expression was downregulated upon IAV infection. Together, our study demonstrated that NUP85 positively regulated IAV infection by interacting with viral PB1 and PB2, which may provide new insight into the process of vRNP nuclear import and a novel target for effective antivirals.

Synthesis, Docking and Antimicrobials Evaluation of Novel Pyrazolotriazines as RNA Polymerase Inhibitors

Aims: Producing novel pyrazolotriazines such as pyrazolo[1,5-a][1,3,5]triazine and pyrazolo[5,1-c][1,2,4]triazine derivatives and evaluate their biological activity as antimicrobial agents followed by the Minimum Inhibitory Concentration (MIC) for the most active compounds. Moreover, study the molecular docking and the RNA polymerase inhibitory activity. Background: Pyrazolotriazine derivatives considered one of the most important heterocyclic compounds due to their broad biological activities. Due to the similarity with the purines and thioguanines, the pyrazolo[1,5-a][1,3,5]triazine and pyrazolo[5,1-c][1,2,4]triazine compounds were used as antimetabolic agents. Moreover, many approved drugs contain pyrazolo[1,5- a][1,3,5]triazine ring systems such as (1882L04 and SB-H02), which confirmed the pharmaceutical applications. The key precursor 5-aminopyrazoles 3 which were firstly synthesized by our research group, were used to prepare the novel pyrazolotriazine derivatives. Objective: This study aimed to synthesize novel bioactive pyrazolo[1,5-a][1,3,5]triazine and pyrazolo[5,1- c][1,2,4]triazine derivatives as antimicrobial agents. Also, the Minimum Inhibitory Concentration (MIC) for the most potent compounds was evaluated. On the other hand, the molecular docking study and the RNA polymerase inhibitory activity were measured. Methods: In this work, the 5-aminopyrazoles 3 were used to synthesize 4-amino-7-(arylamino)pyrazolo[1,5- a][1,3,5]triazine-8-carboxamides 7a-c, 4-amino-7-(arylamino)-2-thioxo-1,2-dihydropyrazolo[1,5-a][1,3,5]-triazine- 8-carboxamides 10a-c and 4-amino-3-cyano-7-(aryllamino)pyrazolo[5,1-c][1,2,4]triazine-8-carboxamides 12ac. The newly resultant compounds were evaluated as antibacterial agents by using (Gram-positive bacteria) such as [Staphylococcus aureus and Streptococcus mutans], and (Gram-negative bacteria) such as [Escherichia coli, Pseudomonas aeruginosa, and Klebsiella]. Moreover, the new compounds were evaluated as antifungal agents by using Candids Albicans fungal strain. Also, the Minimum Inhibitory Concentration (MIC) for the most potent compounds was measured. For all the synthesized compounds, the molecular docking studies were recorded and the RNA polymerase inhibitory activity was measured for the high docking score compounds. Results: The results revealed that most of the prepared compounds such as 7b, 10b, 10c, 12a, 12b, and 12c showed moderate activity towards some of the used strains. The MIC evaluations were recorded for the most active tested compounds 7b, 10b, 10c, 12a and 12c. On the other hand, the most potent and the high docking score compounds (10c, 12a and 12c), were measured in vitro to inhibit RNA polymerase enzyme. Conclusion: A number of novel bioactive pyrazolo[1,5-a][1,3,5]triazine and pyrazolo[5,1-c][1,2,4]triazine derivatives were synthesized. All the resultant compounds were screened for their antimicrobials activity and the MIC test was measured for the most potent compounds. In addition, the in vitro to inhibit RNA polymerase enzyme was evaluated for the most active high docking score compounds. Other: Most of the heterocyclic ring systems have remarkable activities in all fields, especially in pharmaceutical applications.

Long noncoding RNA#45 exerts broad inhibitory effect on influenza a virus replication via its stem ring arms

ABSTRACT A growing body of evidence suggests the pivotal role of long non-coding RNA (lncRNA) in influenza virus infection. Based on next-generation sequencing, we previously demonstrated that Lnc45 was distinctively stimulated by H5N1 influenza virus in mice. In this study, we systematically investigated the specific role of Lnc45 during influenza A virus (IAV) infection. Through qRT-PCR, we first demonstrated that Lnc45 is highly up-regulated by different subtypes of IAV strains, including H5N1, H7N9, and H1N1 viruses. Using RNA-FISH and qRT-PCR, we then found that Lnc45 can translocate from nuclear to cytoplasm during H5N1 virus infection. In addition, forced Lnc45 expression dramatically impeded viral replication of H1N1, H5N1, and H7N9 virus, while abolish of Lnc45 expression by RNA interference favored replication of these viruses, highlighting the potential broad antiviral activity of Lnc45 to IAV. Correspondingly, overexpression of Lnc45 inhibited viral polymerase activity and suppressed IAV-induced cell apoptosis. Moreover, Lnc45 significantly restrained nuclear aggregation of viral NP and PA proteins during H5N1 virus infection. Further functional study revealed that the stem ring arms of Lnc45 mainly mediated the antiviral effect. Therefore, we here demonstrated that Lnc45 functions as a broad-spectrum antiviral factor to inhibit influenza virus replication probably through inhibiting polymerase activity and NP and PA nuclear accumulation via its stem ring arms. Our study not only advances our understanding of the complexity of the IAV pathogenesis but also lays the foundation for developing novel anti-IAV therapeutics targeting the host lncRNA.

In-Silico Screening of Phytochemicals in Febrifuges Used in Siddha Medicine for SARS-CoV-2 RNA Dependent RNA polymerase Inhibitory Activity

In-Silico Screening of Phytochemicals in Febrifuges Used in Siddha Medicine for SARS-CoV-2 RNA Dependent RNA polymerase Inhibitory Activity

1,2,4-Triazolo[1,5-a]pyrimidines as a Novel Class of Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Activity.

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors.

Mycobacterial polymerases: A possible drug target for TB treatment

Mycobacterium tuberculosis causes a deadly disease called Tuberculosis (TB). It enters in a healthy person through respiration and ultimately reaches into lung epithelial cells, where it attacks on lung macrophages to replicate in it and increase its population. For replication process, polymerase activity is essential. There are some compounds which inhibit polymerase activity or denature polymerase enzyme such as Humic acid, Heparin, Urea, Organic compounds, Phenolic compounds etc. In this manuscript, we are trying to show importance of M. tuberculosis pathogenesis by using polymerase activity that may be therapeutic drug target for TB.

Insights into structural dynamics of allosteric binding sites in HCV RNA-dependent RNA polymerase

Inhibition of the viral RNA-dependent RNA polymerase (RdRp) to resolve chronic infection is a useful therapeutic strategy against Hepatitis C virus (HCV). Non-nucleoside inhibitors (NNIs) of RdRp are small molecules that bind tightly with allosteric sites on the enzyme, thereby inhibiting polymerase activity. A large number of crystal structures (176) were studied to establish the structure–activity relationship along with the mechanism of inhibition and resistance between HCV RdRp and NNIs at different allosteric sites. The structure and the associated dynamics are the blueprint to understand the function of the protein. We have implemented the ligand-based pharmacophore and molecular dynamic simulations to extract the possible local and global characteristics of RdRp upon NNI binding and the structural–dynamical features possessed by the known actives. Our results suggest that the NNI binding induces significant fluctuations at the atomic level which are critical for enzymatic activity, with minimal global structural alterations. Residue-wise mapping of interactions of NNIs at different sites exhibited some conserved interaction patterns of key amino acids and water molecules. Here, the structural insights are explored to understand the correlation between the dynamics of protein’s subdomains and function at the molecular level, useful for genotype-specific rational designing of NNIs.Communicated by Ramaswamy H. Sarma

Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using 4-Aminobiphenyl-Modified DNA.

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356–6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

Evaluation of DNA polymerase inhibitory activities of depsidones, depsides and diaryl ethers from the Vietnamese lichen Rimelia clavulifera

AbstractThe Vietnamese lichen Rimelia clavulifera of the family Parmeliaceae was chemically investigated to isolate fourteen known compounds. The inhibitory activities of the isolated lichen substances against mammalian DNA polymerases and human cancer cell growth were assessed. β‐Collatolic acid exhibited inhibitory activities against mammalian DNA polymerases α, β, and κ with IC50 values 9.8 ± 0.6, 8.8 ± 0.5, 19.5 ± 1.1 μM, respectively and showed cytotoxic effects against the HeLa cell line with an IC50 value of 22.2 ± 2.4 μM.

Functionalised bicyclic tetramates derived from cysteine as antibacterial agents.

Routes to bicyclic tetramates derived from cysteine permitting ready incorporation of functionality at two different points around the periphery of a heterocyclic skeleton are reported. This has enabled the identification of systems active against Gram-positive bacteria, some of which show gyrase and RNA polymerase inhibitory activity. In particular, tetramates substituted with glycosyl side chains, chosen to impart polarity and aqueous solubility, show high antibacterial activity coupled with modest gyrase/polymerase activity in two cases. An analysis of physicochemical properties indicates that the antibacterially active tetramates generally occupy physicochemical space with MW of 300-600, clog D7.4 of -2.5 to 4 and rel. PSA of 11-22%. This work demonstrates that biologically active 3D libraries are readily available by manipulation of a tetramate skeleton.

Abstract 5232: The molecular basis for specific recognition of the biologically relevant hybrid-2 type human telomeric G-quadruplex by epiberberine

Abstract G-quadruplex structures have been shown to form in human telomeres, and the formation of G-quadruplexes can inhibit telomerase, which plays a key role in cancers by stabilizing telomere length and integrity thereby granting limitless replicative potential. The human telomeric G-quadruplex is thus considered to be a potential target for cancer therapeutics. In physiologically relevant potassium solution, human telomeric DNA sequences form two equilibrating hybrid-type G-quadruplex structures, with the hybrid-2 structure being the predominant form in extended sequences. We discovered that epiberberine (EPI), a naturally occurring protoberberine alkaloid, can specifically bind the hybrid-2 human telomeric G-quadruplex and can induce the conversion to the hybrid-2 structure. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of the 1:1 complex of EPI and hybrid-2 human telomeric G-quadruplex. Our NMR structure shows EPI bound at the 5’ end of the hybrid-2 telomeric quadruplex with an unexpectedly large drug-induced conformational change in the flanking and loop regions, creating a very well-defined drug binding pocket with extensive capping structures. The EPI molecule and 5’ flanking adenine form an induced quasi-triad plane which is intercalated between the external 5’ tetrad and capping structures. Two layers of new capping structures are formed with the 5’ flanking segment and the second TTA lateral loop to cover the “intercalated quasi-triad”. Notably, the well-defined EPI-induced multi-layer binding-site arrangement is only possible in the hybrid-2 folding topology of telomeric DNA. Our results provide important insights into specific targeting of the physiologically relevant hybrid-2 human telomeric G-quadruplex by a small molecule compound; EPI’s asymmetric crescent-shape and the positioning of its dioxolane moiety, as well as the hybrid-2 folding and the loop and flanking bases in the human telomeric DNA sequence, all contribute toward the specific recognition of EPI. In addition, we have conducted polymerase stop assays, which confirmed that EPI can stabilize the human telomeric G-quadruplex to inhibit polymerase activity. Citation Format: Clement Lin, Guanhui Wu, Yong Shao, Danzhou Yang. The molecular basis for specific recognition of the biologically relevant hybrid-2 type human telomeric G-quadruplex by epiberberine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2017-5232

Purification and optimization of conditions for DNA polymerase isolated from thermophile bacteria Bacillus caldolyticus

Тhermophilic bacteria Bacillus caldolyticus isolated from the hot spring in Bansko, Republic of Macedonia, were used for the isolation of DNA polymerase. Bacterial cells were disrupted by sonication and the first step in the purification of DNA polymerase was 40% ammonium sulfate precipitation. This was followed by chromatographic procedures on Sephadex G-50, DE-52 and CM-52 cellulose. DNA polymerase activity was analyzed at each step of purification using the incorporation of 3H dATP in activated calf thymus DNA. The purity of the DNA polymerase was analyzed on SDS PAGE. Optimal conditions of activity were determined for temperature, pH, dNTP and Mg++ concentration. In addition, the effect of ethanol and EDTA as possible inhibitors of polymerase activity was also analyzed. The optimal temperature of DNA polymerase was 66 ºC; the optimal pH was 7.2, optimal MgCl2 concentration was 2.5 mM, and the optimal substrate concentration was 2.5 × 10–6 M dNTP. The inhibitory effect of EDTA and ethanol on DNA polymerase was above 10 mM and 10%, respectively.

Polyprenylated Benzoylphloroglucinols with DNA Polymerase Inhibitory Activity from the Fruits of Garcinia schomburgkiana.

Chemical investigation of the fruits of Garcinia schomburgkiana collected in Vietnam led to the isolation of eight new schomburgkianones, A-H (1-8), four known (9-12) polyprenylated benzoylphloroglucinols, and four known biflavonoids. The structures of these compounds were elucidated by spectroscopic and chemical means. The absolute configuration at C-40 of 1 and 2 was determined by (1)H NMR analyses of their MPA esters. The configuration of the bicyclo[3.3.1]nonane core of the polyprenylated benzoylphloroglucinols was assigned by comparison of their experimental ECD spectra with those of related compounds. The polyprenylated benzoylphloroglucinols exhibited inhibitory activities against mammalian DNA polymerases α and λ, with IC50 values ranging from 5.0 to 8.8 μM. Compounds 1, 2, 4, 5, and 9-11 showed cytotoxic effects against HeLa human cervical cancer cells with median lethal dose values lower than 10 μM.

Interaction of polycationic Ni(II)-salophen complexes with G-quadruplex DNA.

A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 μM for G-quadruplexes and greater than 2 μM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.