Redox-Specific Allosteric Modulation of the Conformational Dynamics of κB DNA by Pirin in the NF-κB Supramolecular Complex.

The molecular basis of gene regulation by Nuclear Factor-κB (NF-κB) transcription factors and their coregulators is not well understood. This family of transcription factors controls a number of essential subcellular processes. Human Pirin, a nonheme iron (Fe) binding protein, has been shown to modulate the binding affinity between p65 homodimeric NF-κB and κB DNA. However, the allosteric effect of the active Fe(III) form of Pirin on the DNA has not been established. Here, we use multiple microsecond-long molecular dynamics simulations to explore the conformational dynamics of the free DNA, the p65-DNA complex, and the Pirin-p65-DNA supramolecular complex. We show that only the Fe(III) form of Pirin enhances the affinity between p65 and the DNA in the Pirin-p65-DNA supramolecular complex, in agreement with experiments. Additionally, the results provide atomistic details of the effect of the active Fe(III) form of Pirin on the DNA upon binding to the p65-DNA complex. In general, unlike the Fe(II) form of Pirin, binding of the Fe(III) form of Pirin to the p65-DNA complex significantly alters both the conformational dynamics of the DNA and the interactions between p65 and the DNA. The results provide atomic level understanding of the modulation of the DNA as a result of a redox-specific Fe(II)/Fe(III) coregulation of NF-κB by Pirin, knowledge that is necessary to fully understand normal and aberrant subcellular processes and the role of a subtle single electron redox process in gene regulation.

SRI-32743, a novel allosteric modulator, attenuates HIV-1 Tat protein-induced inhibition of the dopamine transporter and alleviates the potentiation of cocaine reward in HIV-1 Tat transgenic mice.

Cocaine abuse increases the incidence of HIV-1-associated neurocognitive disorders. We have demonstrated that HIV-1 transactivator of transcription (Tat) allosterically modulates dopamine (DA) reuptake through the human DA transporter (hDAT), potentially contributing to Tat-induced cognitive impairment and potentiation of cocaine conditioned place preference (CPP). This study determined the effects of a novel allosteric modulator of DAT, SRI-32743, on the interactions of HIV-1 Tat, DA, cocaine, and [3H]WIN35,428 with hDAT in vitro. SRI-32743 (50 nM) attenuated Tat-induced inhibition of [3H]DA uptake and decreased the cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting a SRI-32743-mediated allosteric modulation of the Tat-DAT interaction. In further in vivo studies utilizing doxycycline-inducible Tat transgenic (iTat-tg) mice, 14 days of Tat expression significantly reduced the recognition index by 31.7% in the final phase of novel object recognition (NOR) and potentiated cocaine-CPP 2.7-fold compared to responses of vehicle-treated control iTat-tg mice. The Tat-induced NOR deficits and potentiation of cocaine-CPP were not observed in saline-treated iTat-tg or doxycycline-treated G-tg (Tat-null) mice. Systemic administration (i.p.) of SRI-32743 prior to behavioral testing ameliorated Tat-induced impairment of NOR (at a dose of 10 mg/kg) and the Tat-induced potentiation of cocaine-CPP (at doses of 1 or 10 mg/kg). These findings demonstrate that Tat and cocaine interactions with DAT may be regulated by compounds interacting at the DAT allosteric modulatory sites, suggesting a potential therapeutic intervention for HIV-infected patients with concurrent cocaine abuse.

Binding Mode of Human Norepinephrine Transporter Interacting with HIV-1 Tat.

The increase of HIV infection in macrophages results in HIV proteins being released, like HIV Tat which impairs the function of monoamine transporters. HIV-infected patients have displayed increased synaptic levels of dopamine (DA) due to reduced binding and function of monoamine transporters such as the norepinephrine transporter (NET) and the dopamine transporter (DAT). Development of a three-dimensional model of the HIV-1 Tat-human NET (hNET) binding complex would help reveal how HIV-1 Tat causes toxicity in the neuron by affecting DA uptake. Here we use computational techniques such as molecular modeling to study microscopic properties and molecular dynamics of the HIV-1 Tat-hNET binding. These modeling techniques allow us to analyze noncovalent interactions and observe residue-residue contacts to verify a model structure. The modeling results studied here show that HIV-1 Tat-hNET binding is highly dynamic and that HIV-1 Tat preferentially binds to hNET in its outward-open state. In particular, HIV-1 Tat forms hydrogen bond interactions with side chains of hNET residues Y84, K88, and T544. The favorable hydrogen bonding interactions of HIV-1 Tat with the hNET side chain residues Y84 and T544 have been validated by our subsequently performed DA uptake activity assays and site-directed mutagenesis, suggesting that the modeled HIV-1 Tat-hNET binding mode is reasonable. These mechanistic and structural insights gained through homology models discussed in this study are expected to encourage the pursuit of pharmacological and biochemical studies on HIV-1 Tat interacting with hNET mechanisms and detailed structures.

Epigenetic Regulation of Wnt Signaling by Carboxamide-Substituted Benzhydryl Amines that Function as Histone Demethylase Inhibitors.

Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (ß-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity.