A microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA complex by doxorubicin.

The transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription activator that binds to GC-rich recognition sites in a number of essential cellular and viral promoters. In addition, direct interference of Sp1 binding to DNA cognate sites using DNA-interacting compounds may provide promising therapies for suppression of cancer progression and viral replication. In this study, we present a rapid, sensitive and cost-effective evaluation of a GC intercalative drug, doxorubicin (DOX), in dissociating the Sp1-DNA complex using fluorescence correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay miniaturization without compromising sensitivity, making it an ideal analytical method for integration of binding assays into high-throughput, microfluidic platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing network is used to achieve specific drug concentrations for drug titration experiments. Using FCS measurements, the IC50 of DOX on the dissociation of Sp1-DNA complex is estimated to be 0.55 microM, which is comparable to that measured by the electrophoretic mobility shift assay (EMSA). However, completion of one drug titration experiment on the proposed microfluidic-FCS platform is accomplished using only picograms of protein and DNA samples and less than 1 h total assay time, demonstrating vast improvements over traditional ensemble techniques.

Inhibition of HSV-1 replication and reactivation by the mutation-insensitive transcription inhibitor tetra-O-glycyl-nordihydroguaiaretic acid.

Methylated derivatives of nordihydroguaiaretic acid (NDGA)were previously shown to be potent mutation-resistant inhibitors of herpes simplex virus type 1 (HSV-1) which target Sp1 protein binding to critical viral promoters. The hydrophobic nature of these agents, however, renders them relatively water-insoluble and, therefore, limits their applicability. We report here on the anti-HSV-1 properties of a related but water-soluble glycylated derivative of NDGA, tetra-O-glycyl-NDGA (G(4)N). In yield reduction assays, G(4)N inhibited replication of laboratory and clinical strains of wild type HSV-1 and ACV-resistant (HSV-1(R)) strains of HSV-1 in a dose-dependent manner, with average IC(50) values of 4.7 and 3.2 microM against wild-type and HSV-1(R) strains, respectively. An MTT-based cytotoxicity assay revealed a TC(50) value of 73.2 microM for G(4)N on Vero cells, with no reduction in viability detected at concentrations below 30 microM. Similar to its methylated counterparts, G(4)N was found to inhibit transcription of the HSV-1 ICP4 gene, a major immediate early viral regulator, and gel mobility shift assays showed it can block Sp1 protein binding to cognate sites on the ICP4 promoter. In anticipation of its potential use as a systemic anti-HSV-1 agent, we tested G(4)N in a murine trigeminal ganglia (TG) explant model system, and found G(4)N was able to prevent HSV-1 reactivation from explanted and cultured latently infected TG.

Novel antiviral agent tetraglycylated nordihydroguaiaretic acid hydrochloride as a host-dependent viral inhibitor.

A water soluble derivative of nordihydroguaiaretic acid (NDGA), G(4)N (2), synthesized by reaction of NDGA (1) with N,N-dimethylglycine in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine and then with HCl(g) (Scheme 1), competes effectively with the DNA binding domain of recombinant Sp1 protein for binding to the human immunodeficiency virus (HIV) LTR as demonstrated by an electrophoretic mobility-shift assay (EMSA). By blocking Sp1 binding to the HIV LTR, G(4)N suppresses Sp1-regulated HIV Tat transactivation and replication in cultured cells with an IC(50) of 12 microM similar to that of 3'-O-methyl-NDGA as we have previously reported. In addition simian immunodeficiency virus (SIV) replication was completely inhibited by G(4)N at 5.0 microM. G(4)N showed no toxic effect to 174 x CEM cells and H9 cells at 100 microM.