In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses.

The Dengue virus protease NS2B3 cleaves cyclic GMP-AMP synthase to suppress cGAS activation.

Dengue virus (DENV) is one of the most prevalent mosquito-transmitted human viruses that causes significant morbidity and mortality worldwide. To persist in the cell and consequently cause disease, DENV is evolved with mechanisms to suppress the induction of type I interferons by antagonizing cGAS-STING signaling. Using recombinant proteins and in vitro cleavage assays, we have shown that the DENV protease NS2B3 is capable of cleaving cGAS in the N-terminal region without disrupting the C-terminal catalytic center.

Structural basis of higher order oligomerization of KSHV inhibitor of cGAS.

Kaposi's sarcoma-associated herpesvirus (KSHV) inhibitor of cyclic GMP-AMP synthase (cGAS) (KicGAS) encoded by ORF52 is a conserved major tegument protein of KSHV and the first reported viral inhibitor of cGAS. In our previous study, we found that KicGAS is highly oligomerized in solution and that oligomerization is required for its cooperative DNA binding and for inhibiting DNA-induced phase separation and activation of cGAS. However, how KicGAS oligomerizes remained unclear.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies.

Evasion of Intracellular DNA Sensing by Human Herpesviruses.

Sensing of viral constituents is the first and critical step in the host innate immune defense against viruses. In mammalian cells, there are a variety of pathogen recognition receptors (PRRs) that detect diverse pathogen-associated molecular patterns (PAMPs) including viral RNA and DNA. In the past decade, a number of host DNA sensors have been discovered and the underlying sensing mechanisms have been elucidated.

Influence of Flexible "ω" on the Activity of E. coli RNA Polymerase: A Thermodynamic Analysis.

The Escherichia coli RNA polymerase (RNAP) is a multisubunit protein complex containing the smallest subunit, ω. Despite the evolutionary conservation of ω and its role in assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of RNAP with the global chaperone protein GroEL.

A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins, hemoglobin and myoglobin.

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21 ± .

Recent Advances in Nucleic Acid Binding Aspects of Berberine Analogs and Implications for Drug Design.

Berberine is one of the most widely known alkaloids belonging to the protoberberine group exhibiting myriad therapeutic properties. The anticancer potency of berberine appears to derive from its multiple actions including strong interaction with nucleic acids exhibiting adenine-thymine base pair specificity, inhibition of the enzymes topoisomerases and telomerases, and stabilizing the quadruplex structures. It was realized that the development of berberine as a potential anticancer agent necessitates enhancing its nucleic acid binding efficacy through appropriate structural modifications.

Recognition of human telomeric G-quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety.

G-quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13-position with human telomeric G-quadruplex DNA sequence has been investigated by biophysical techniques.

Stepwise unfolding of bovine and human serum albumin by an anionic surfactant: an investigation using the proton transfer probe norharmane.

Interactions of the anionic surfactant sodium dodecyl sulfate (SDS) with the transport proteins bovine serum albumin (BSA) and human serum albumin (HSA) have been divulged using an external photoinduced proton transfer probe, norharmane (NHM). Steady-state fluorometry, time-resolved measurements, micropolarity analysis, circular dichroism (CD), and isothermal titration calorimetry (ITC) have been exploited for the study. With the gradual addition of SDS to the probe-bound proteins, the fluorometric responses of the different prototropic species of NHM exhibit an opposite pattern as to that observed while NHM binds to the proteins.

Spectroscopic studies on the binding interaction of novel 13-phenylalkyl analogs of the natural alkaloid berberine to nucleic acid triplexes.

In this study we have characterized the capability of six 13-phenylalkyl analogs of berberine to stabilize nucleic acid triplex structures, poly(rA)â‹…2poly(rU) and poly(dA)â‹…2poly(dT). Berberine analogs bind to the RNA and DNA triplexes non-cooperatively. As the chain length of the substitution increased beyond CH2, the affinity enhanced up to critical length of (CH2)4, there after which the binding affinity decreased for both the triplexes.

The benzophenanthridine alkaloid chelerythrine binds to DNA by intercalation: photophysical aspects and thermodynamic results of iminium versus alkanolamine interaction.

The interaction of the natural benzophenanthridine alkaloid chelerythrine with DNA was studied by spectroscopy, viscometry and calorimetry techniques. The absorbance and fluorescence properties of the alkaloid were remarkably modified upon binding to DNA and the interaction was found to be cooperative. The mode of binding was principally by intercalation as revealed from viscosity studies and supported from fluorescence quenching, and polarization results.

Interaction of 9-O-(ω-amino) alkyl ether berberine analogs with poly(dT)·poly(dA)*poly(dT) triplex and poly(dA)·poly(dT) duplex: a comparative study.

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices.

Biophysical characterization of the strong stabilization of the RNA triplex poly(U)•poly(A)*poly(U) by 9-O-(ω-amino) alkyl ether berberine analogs.

Background: Binding of two 9-O-(ω-amino) alkyl ether berberine analogs BC1 and BC2 to the RNA triplex poly(U)(•)poly(A)(*)poly(U) was studied by various biophysical techniques.

Methodology/principal Findings: Berberine analogs bind to the RNA triplex non-cooperatively. The affinity of binding was remarkably high by about 5 and 15 times, respectively, for BC1 and BC2 compared to berberine.

Biophysical studies on the effect of the 13 position substitution of the anticancer alkaloid berberine on its DNA binding.

The structural effects and thermodynamics of the DNA binding of six berberine analogues with alkyl chains of varying length and a terminal phenyl group at the C-13 position were investigated. All the analogues bound DNA noncooperatively in contrast to the cooperative binding of berberine. The binding affinity was higher and the effect of the chain length was only up to (CH(2))(3), after which the binding affinity decreased slightly.