Inthevirion coreofretroviruses, thegenomic RNAis tightly associated withnucleocapsid (NC)protein molecules, forming thenucleocapsid structure. NC protein, ahighly basic protein withtwozincfingers, isindispensable forRNAdimerization, encapsidation andtheinitiation ofreverse transcription inavian, murine andhumanretroviruses. Hereweshowthat NC protein ofHIV-1 (NCp7) andNCp7mutants bindtoDNA fragments representing proviral DNA sequences, forming stable complexes. NCp7andNCp7mutants formhighmolecular weight complexes withlarge DNA fragments andshowcooperativity inbinding tothe DNAs.Itappears thattheconserved basicresidues, andnotthezincfingers, areimportant forcomplex formation. Inaddition, NCp7andseveral NCp7mutants protect DNAsfromnuclease digestion while theDNA endsappear tobepoorly protected. NCp7hasbeen foundtobindtostrong stopcDNA,theinitial product ofreverse transcription, andtopromote theannealing ofthis cDNAtoHIV-1 RNAcorresponding tothe3'end ofthegenome.Inaddition, NCp7slightly stimulates an invitro INcleavage assay. Theseresults indicate that theinteractions between NCp7andproviral DNAmay beimportant during provirus synthesis and/or prior to integration.

Interactions between HIV-1nucleocapsid protein andviral DNA may haveimportant functions intheviral life cycle

NCp7: targeting a multitasking protein for next-generation anti-HIV drug development part 1: covalent inhibitors.

HIV/AIDS is still one of the leading causes of death worldwide. Current drugs that target the canonical steps of the HIV-1 life cycle are efficient in blocking viral replication but are unable to eradicate HIV-1 from infected patients. Moreover, drug resistance (DR) is often associated with the clinical use of these molecules, thus raising the need for novel drug candidates as well as novel putative drug targets. In this respect, pharmacological inhibition of the highly conserved and multifunctional nucleocapsid protein (NC) of HIV-1 is considered a promising alternative to current drugs, particularly to overcome DR. Here, using a multidisciplinary approach combining in silico screening, fluorescence-based molecular assays, and cellular antiviral assays, we identified nordihydroguaiaretic acid (6), as a novel natural product inhibitor of NC. By using NMR, mass spectrometry, fluorescence spectroscopy, and molecular modeling, 6 was found to act through a dual mechanism of action never highlighted before for NC inhibitors (NCIs). First, the molecule recognizes and binds NC noncovalently, which results in the inhibition of the nucleic acid chaperone properties of NC. In a second step, chemical oxidation of 6 induces a potent chemical inactivation of the protein. Overall, 6 inhibits NC and the replication of wild-type and drug-resistant HIV-1 strains in the low micromolar range with moderate cytotoxicity that makes it a profitable tool compound as well as a good starting point for the development of pharmacologically relevant NCIs.

Structure-Based Identification of HIV-1 Nucleocapsid Protein Inhibitors Active against Wild-Type and Drug-Resistant HIV-1 Strains

Nucleocapsid protein 7 (NCp7) is an attractive target for anti-HIV drug development. Here we found that ruthenium complexes are reactive to NCp7 and various Ru-agents exhibit significantly different reactivity. Interestingly, the zinc-finger domains of NCp7 also demonstrate different affinity to Ru-complexes; the C-terminal domain is much more reactive than the N-terminal domain. Each zinc-finger domain of NCp7 binds up to three Ru-motifs, and the ruthenium binding causes zinc-ejection from NCp7 and disrupts the protein folding. Therefore, ruthenium complexes interfere with the DNA binding of NCp7 and interrupt the protein function. The different reactivity of Ru-agents suggests a feasible strategy for improving the targeting of NCp7 by ligand design. This work provides an insight into the mechanism of ruthenium complex with NCp7, and suggests more potential application of ruthenium drugs.

Selective Targeting of the Zinc Finger Domain of HIV Nucleocapsid Protein NCp7 with Ruthenium Complexes.

Small molecule inhibitors of the HIV-1 nucleocapsid protein (NC) are considered as promising agents in the treatment of HIV/AIDS. In an effort to exploit the privileged 2-amino-4-phenylthiazole moiety in NC inhibition, here we conceived, synthesized, and tested in vitro 18 NC inhibitors (NCIs) bearing a double functionalization. In these NCIs, one part of the molecule is deputed to interact noncovalently with the NC hydrophobic pocket, while the second portion is designed to interact with the N-terminal domain of NC. This binding hypothesis was verified by molecular dynamics simulations, while the linkage between these two pharmacophores was found to enhance antiretroviral activity both on the wild-type virus and on HIV-1 strains with resistance to currently licensed drugs. The two most interesting compounds 6 and 13 showed no cytotoxicity, thus becoming valuable leads for further investigations.

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K. George Rosenker

Papers

Preclinical evaluation of a mercaptobenzamide and its prodrug for NCp7-targeted inhibition of human immunodeficiency virus.