Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro

被引:192
作者
Bacha, U [1 ]
Barrila, J [1 ]
Velazquez-Campoy, A [1 ]
Leavitt, SA [1 ]
Freire, E [1 ]
机构
[1] Johns Hopkins Univ, Dept Biol, Baltimore, MD 21218 USA
关键词
D O I
10.1021/bi0361766
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the (alpha-helical domain. Differential scanning calorimetry indicates that the (alpha-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid., This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.
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页码:4906 / 4912
页数:7
相关论文
共 28 条
  • [1] Inhibition of a model protease -: pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa
    Alur, HH
    Desai, RP
    Mitra, AK
    Johnston, TP
    [J]. INTERNATIONAL JOURNAL OF PHARMACEUTICS, 2001, 212 (02) : 171 - 176
  • [2] Coronavirus main proteinase (3CLpro) structure:: Basis for design of anti-SARS drugs
    Anand, K
    Ziebuhr, J
    Wadhwani, P
    Mesters, JR
    Hilgenfeld, R
    [J]. SCIENCE, 2003, 300 (5626) : 1763 - 1767
  • [3] Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α-helical domain
    Anand, K
    Palm, GJ
    Mesters, JR
    Siddell, SG
    Ziebuhr, J
    Hilgenfeld, R
    [J]. EMBO JOURNAL, 2002, 21 (13) : 3213 - 3224
  • [4] Copeland R. A., 2004, ENZYMES PRACTICAL IN
  • [5] Kinetic properties of saquinavir-resistant mutants of human immunodeficiency virus type 1 protease and their implications in drug resistance in vivo
    Ermolieff, J
    Lin, XL
    Tang, J
    [J]. BIOCHEMISTRY, 1997, 36 (40) : 12364 - 12370
  • [6] Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase
    Fan, KQ
    Wei, P
    Feng, Q
    Chen, SD
    Huang, CK
    Ma, L
    Lai, B
    Pei, JF
    Liu, Y
    Chen, JG
    Lai, LH
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2004, 279 (03) : 1637 - 1642
  • [7] Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites
    Fehér, A
    Weber, IT
    Bagossi, P
    Boross, P
    Mahalingam, B
    Louis, JM
    Copeland, TD
    Torshin, IY
    Harrison, RW
    Tözsér, J
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 2002, 269 (16): : 4114 - 4120
  • [8] KINETIC CHARACTERIZATION AND CROSS-RESISTANCE PATTERNS OF HIV-1 PROTEASE MUTANTS SELECTED UNDER DRUG PRESSURE
    GULNIK, SV
    SUVOROV, LI
    LIU, BS
    YU, B
    ANDERSON, B
    MITSUYA, H
    ERICKSON, JW
    [J]. BIOCHEMISTRY, 1995, 34 (29) : 9282 - 9287
  • [9] CORONAVIRUS PROTEIN PROCESSING AND RNA-SYNTHESIS IS INHIBITED BY THE CYSTEINE PROTEINASE-INHIBITOR E64D
    KIM, JC
    SPENCE, RA
    CURRIER, PF
    LU, XT
    DENISON, MR
    [J]. VIROLOGY, 1995, 208 (01) : 1 - 8
  • [10] LICALSI C, 1991, J BIOL CHEM, V266, P19519