A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

Functional and structural characterization of 2-amino-4-phenylthiazole inhibitors of the HIV-1 nucleocapsid protein with antiviral activity.

The nucleocapsid protein (NC) is a highly conserved protein in diverse HIV-1 subtypes that plays a central role in virus replication, mainly by interacting with conserved nucleic acid sequences. NC is considered a highly profitable drug target to inhibit multiple steps in the HIV-1 life cycle with just one compound, a unique property not shown by any of the other antiretroviral classes. However, most of NC inhibitors developed so far act through an unspecific and potentially toxic mechanism (zinc ejection) and are mainly being investigated as topical microbicides. In an effort to provide specific NC inhibitors that compete for the binding of nucleic acids to NC, here we combined molecular modeling, organic synthesis, biophysical studies, NMR spectroscopy, and antiviral assays to design, synthesize, and characterize an efficient NC inhibitor endowed with antiviral activity in vitro, a desirable property for the development of efficient antiretroviral lead compounds.

Characterization of the inhibition mechanism of HIV-1 nucleocapsid protein chaperone activities by methylated oligoribonucleotides.

Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC.

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5' thymine interacts with residues of the N-terminal zinc knuckle and the 3' guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA-protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids.

How the HIV-1 nucleocapsid protein binds and destabilises the (-)primer binding site during reverse transcription.

The human immunodeficiency virus type 1 nucleocapsid protein (NCp7) plays an important role in the second strand transfer during reverse transcription. It promotes annealing of the 18-nucleotide complementary DNA primer-binding site (PBS) sequences at the 3' ends of (-)DNA and (+)DNA. NMR studies show that NCp7(12-55) and NCp7(1-55) interact at the 5' end of the loop of DeltaP(-)PBS, a (-)PBS derivative without the 3' protruding sequence, in a slow-exchange equilibrium. This interaction is mediated through the binding of the hydrophobic plateau (Val13, Phe16, Thr24, Ala25, Trp37, and Met46) on the zinc finger domain of both peptides to the 5-CTG-7 sequence of DeltaP(-)PBS. The stacking of the Trp37 aromatic ring with the G7 residue likely constitutes the determinant factor of the interaction. Although NCp7(12-55) does not melt the DeltaP(-)PBS stem-loop structure, it opens the loop and weakens the C5.G11 base pair next to the loop. Moreover, NCp7(12-55) was also found to bind but with lower affinity to the 10-CGG-12 sequence in an intermediate-exchange equilibrium on the NMR time scale. The loop modifications may favour a kissing interaction with the complementary (+)PBS loop. Moreover, the weakening of the upper base pair of the stem likely promotes the melting of the stem that is required to convert the kissing complex into the final (+/-)PBS extended duplex.