Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen.
BACKGROUND
HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex.


RESULTS
Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades.


CONCLUSIONS
Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
Ronald P
RP
Trible
Purushottam
P
Narute
Lori A
LA
Emert-Sedlak
John Jeff
JJ
Alvarado
Katelyn
K
Atkins
Laurel
L
Thomas
Toshiaki
T
Kodama
Naveena
N
Yanamala
Vasiliy
V
Korotchenko
Billy W
BW
Day
Gary
G
Thomas
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA. tsmithga@pitt.edu.
Thomas E
TE
Smithgall
D019380:Anti-HIV Agents / Q000302:isolation & purification / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D062105:Molecular Docking Simulation; D011485:Protein Binding / Q000187:drug effects; D051572:Proto-Oncogene Proteins c-hck / Q000037:antagonists & inhibitors* / Q000235:genetics; D011810:Quinoxalines / Q000302:isolation & purification / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D013449:Sulfonamides / Q000302:isolation & purification / Q000494:pharmacology*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors* / Q000235:genetics; D000096926:Benzenesulfonamides
D019380:Anti-HIV Agents; D051572:Proto-Oncogene Proteins c-hck; D011810:Quinoxalines; D013449:Sulfonamides; D054311:nef Gene Products, Human Immunodeficiency Virus; D000096926:Benzenesulfonamides; C514837:nef protein, Human immunodeficiency virus 1; C496631:HCK protein, human