Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action.
The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.
Basrur
V
V
Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Song
Y
Y
Mazur
S J
SJ
Higashimoto
Y
Y
Turpin
J A
JA
Rice
W G
WG
Inman
J K
JK
Appella
E
E
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000494:pharmacology*; D002213:Capsid / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011726:Pyridinium Compounds / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D018629:Spectrometry, Mass, Secondary Ion; D013409:Sulfacetamide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D013450:Sulfones / Q000737:chemistry / Q000494:pharmacology*; D014764:Viral Proteins; D015032:Zinc / Q000032:analysis; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D019380:Anti-HIV Agents; D036022:Capsid Proteins; D015683:Gene Products, gag; C409464:N-(2-(5-pyridiniovaleroylthio)benzoyl)-4-(4-nitrophenylsulfonyl)aniline; C409463:N-(2-(5-pyridiniovaleroylthio)benzoyl)sulfacetamide; C071884:NCP7 protein, Human immunodeficiency virus 1; D011726:Pyridinium Compounds; D011994:Recombinant Proteins; D013450:Sulfones; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D013409:Sulfacetamide; D015032:Zinc