PDBsum entry 2az5

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Cytokine PDB id
Protein chains
132 a.a. *
140 a.a. *
307 ×2
Waters ×226
* Residue conservation analysis
PDB id:
Name: Cytokine
Title: Crystal structure of tnf-alpha with a small molecule inhibitor
Structure: Tumor necrosis factor (tnf-alpha) (tumor necrosis factor ligand superfamily member 2) (tnf-a) (cachectin) [contains: tumor necrosis factor, membrane form. Tumor necrosis factor, soluble form]. Chain: a, b, c, d. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: tnf, tnfa, tnfsf2. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Dimer (from PQS)
2.10Å     R-factor:   0.227     R-free:   0.278
Authors: M.M.He
Key ref:
M.M.He et al. (2005). Small-molecule inhibition of TNF-alpha. Science, 310, 1022-1025. PubMed id: 16284179 DOI: 10.1126/science.1116304
09-Sep-05     Release date:   29-Nov-05    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P01375  (TNFA_HUMAN) -  Tumor necrosis factor
233 a.a.
132 a.a.
Protein chains
Pfam   ArchSchema ?
P01375  (TNFA_HUMAN) -  Tumor necrosis factor
233 a.a.
140 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   1 term 
  Biological process     immune response   1 term 
  Biochemical function     tumor necrosis factor receptor binding     1 term  


DOI no: 10.1126/science.1116304 Science 310:1022-1025 (2005)
PubMed id: 16284179  
Small-molecule inhibition of TNF-alpha.
M.M.He, A.S.Smith, J.D.Oslob, W.M.Flanagan, A.C.Braisted, A.Whitty, M.T.Cancilla, J.Wang, A.A.Lugovskoy, J.C.Yoburn, A.D.Fung, G.Farrington, J.K.Eldredge, E.S.Day, L.A.Cruz, T.G.Cachero, S.K.Miller, J.E.Friedman, I.C.Choong, B.C.Cunningham.
We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.
  Selected figure(s)  
Figure 1.
Fig. 1. (A) Chemical structure of the small molecule TNF- inhibitor. See (12) for synthetic route used. (B) Compound inhibition of TNF- binding to TNFR1 in vitro. An ELISA (12) was used to measure inhibition of solution-phase TNF-R1 horseradish peroxidase conjugate binding to biotinylated TNF- immobilized on a strepavidin-coated microtiter plate by serial dilutions of compound. The solid line represents a four-parameter curve fit (20) that yielded an IC[50] value of 22 µM. (C) Compound inhibition of TNF- induced I B- depletion in HeLa cells. Cells were treated with sufficient TNF- or IL-1ß to give an 80% of maximal I B- depletion response after a 30-min exposure (0.4 and 0.04 ng/ml, respectively), as measured in an assay of cell lysates (12). Solid circles show that compound addition inhibits this TNF- -induced I B- depletion and yields an IC[50] value of 4.6 µM. Open circles show that compound addition does not affect orthogonal IL-1ß-induced I B- depletion. Error bars indicate standard deviations for triplicate measurements.
Figure 4.
Fig. 4. Data showing compound-induced formation of TNF- dimer in solution. (A) Detection of TNF- oligomeric state by mass spectrometry. Spectrum is a deconvoluted neutral scale mass spectrum of noncovalent TNF- trimer complex visualized as three peaks due to plus and minus N-terminal methionine heterogeneity of TNF- sample. The observed masses of 52,052, 52,182, and 52,312 daltons corresponds to the trimer complex containing either zero, one, or two subunits with N-terminal methionines, respectively. (B) Analogous spectrum of 10 µM TNF- incubated with 100 µM SP307. Masses at 34,704 and 35,252 correspond to noncovalent complex of TNF- dimer and TNF- dimer-compound, respectively. (C) Increase in TNF- exchangeable hydrogens induced by compound. Plot shows time course for hydrogen-deuterium exchange of 1 µM TNF- dissolved into either D[2]O alone (open circles) or D[2]O plus 30 µM compound (solid circles). Procedure was performed as described (12). Calculations of exposed surface area predict 88, 99, and 110 exchangeable hydrogens for each subunit within the TNF- trimer, dimer, and monomer structures, respectively (21, 22). (D) TNF- subunit dissociation rates in the presence and absence of compound. The relief of fluorescence homoquenching of 100 nM T7C-AF TNF- after dilution in a 200-fold excess of unlabeled TNF- was used to monitor subunit disassociation from TNF- trimer (solid circles). Nonlinear regression analysis using the appropriate kinetics equation (12) gave a calculated rate of 0.000093 s-1 per monomer dissociation event (solid circles). Addition of 30 µM compound to the homoquenching assay accelerates the observed time course of fluorescence increase and yields a calculated rate constant of 0.059 s-1 per monomer dissociation event (solid triangles). Open circles show fluorescence in the absence of added unlabeled TNF- . Error bars represent the standard deviation of triplicate measurements.
  The above figures are reprinted by permission from the AAAs: Science (2005, 310, 1022-1025) copyright 2005.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
23103871 B.Kim, A.Eggel, S.S.Tarchevskaya, M.Vogel, H.Prinz, and T.S.Jardetzky (2012).
Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor.
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Structure-based repurposing of FDA-approved drugs as TNF-α inhibitors.
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20489699 D.Faustman, and M.Davis (2010).
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20117133 J.L.Galzi, M.Hachet-Haas, D.Bonnet, F.Daubeuf, S.Lecat, M.Hibert, J.Haiech, and N.Frossard (2010).
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Thermodynamic additivity of sequence variations: an algorithm for creating high affinity peptides without large libraries or structural information.
  PLoS One, 5, e15432.  
20979208 P.Buchwald (2010).
Small-molecule protein-protein interaction inhibitors: therapeutic potential in light of molecular size, chemical space, and ligand binding efficiency considerations.
  IUBMB Life, 62, 724-731.  
20813201 T.K.Khera, A.D.Dick, and L.B.Nicholson (2010).
Mechanisms of TNFα regulation in uveitis: focus on RNA-binding proteins.
  Prog Retin Eye Res, 29, 610-621.  
20421509 V.N.Malashkevich, N.G.Dulyaninova, U.A.Ramagopal, M.A.Liriano, K.M.Varney, D.Knight, M.Brenowitz, D.J.Weber, S.C.Almo, and A.R.Bresnick (2010).
Phenothiazines inhibit S100A4 function by inducing protein oligomerization.
  Proc Natl Acad Sci U S A, 107, 8605-8610.
PDB code: 3ko0
19641779 B.A.Shah, R.Chib, P.Gupta, V.K.Sethi, S.Koul, S.S.Andotra, A.Nargotra, S.Sharma, A.Pandey, S.Bani, B.Purnima, and S.C.Taneja (2009).
Saponins as novel TNF-alpha inhibitors: isolation of saponins and a nor-pseudoguaianolide from Parthenium hysterophorus.
  Org Biomol Chem, 7, 3230-3235.  
19007309 C.Ottmann, M.Weyand, A.Wolf, J.Kuhlmann, and C.Ottmann (2009).
Applicability of superfolder YFP bimolecular fluorescence complementation in vitro.
  Biol Chem, 390, 81-90.
PDB code: 3ed8
19875081 F.Buller, Y.Zhang, J.Scheuermann, J.Schäfer, P.Bühlmann, and D.Neri (2009).
Discovery of TNF inhibitors from a DNA-encoded chemical library based on diels-alder cycloaddition.
  Chem Biol, 16, 1075-1086.  
19812033 S.H.Lawrence, U.D.Ramirez, T.Selwood, L.Stith, and E.K.Jaffe (2009).
Allosteric inhibition of human porphobilinogen synthase.
  J Biol Chem, 284, 35807-35817.  
19274442 Y.Cao, Z.Wang, X.Bu, S.Tang, Z.Mei, and P.Liu (2009).
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18502680 A.A.Kruglov, A.Kuchmiy, S.I.Grivennikov, A.V.Tumanov, D.V.Kuprash, and S.A.Nedospasov (2008).
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  Cytokine Growth Factor Rev, 19, 231-244.  
18694376 F.Fantuzzi, M.Del Giglio, P.Gisondi, and G.Girolomoni (2008).
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  Expert Opin Ther Targets, 12, 1085-1096.  
  18582039 L.L.Chan, M.Pineda, J.T.Heeres, P.J.Hergenrother, and B.T.Cunningham (2008).
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  18925972 M.K.McCoy, and M.G.Tansey (2008).
TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease.
  J Neuroinflammation, 5, 45.  
17916444 M.Wong, D.Ziring, Y.Korin, S.Desai, S.Kim, J.Lin, D.Gjertson, J.Braun, E.Reed, and R.R.Singh (2008).
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  Clin Immunol, 126, 121-136.  
18936751 R.B.Russell, and P.Aloy (2008).
Targeting and tinkering with interaction networks.
  Nat Chem Biol, 4, 666-673.  
19578473 S.H.Lawrence, and E.K.Jaffe (2008).
Expanding the Concepts in Protein Structure-Function Relationships and Enzyme Kinetics: Teaching using Morpheeins.
  Biochem Mol Biol Educ, 36, 274-283.  
18559269 S.H.Lawrence, U.D.Ramirez, L.Tang, F.Fazliyez, L.Kundrat, G.D.Markham, and E.K.Jaffe (2008).
Shape shifting leads to small-molecule allosteric drug discovery.
  Chem Biol, 15, 586-596.  
18438977 U.A.Mirza, G.Chen, Y.H.Liu, R.J.Doll, V.M.Girijavallabhan, A.K.Ganguly, and B.N.Pramanik (2008).
Mass spectrometric studies of potent inhibitors of farnesyl protein transferase--detection of pentameric noncovalent complexes.
  J Mass Spectrom, 43, 1393-1401.  
18378892 Z.Zhou, X.Song, A.Berezov, G.Zhang, Y.Li, H.Zhang, R.Murali, B.Li, and M.I.Greene (2008).
Human glucocorticoid-induced TNF receptor ligand regulates its signaling activity through multiple oligomerization states.
  Proc Natl Acad Sci U S A, 105, 5465-5470.  
18075579 J.A.Wells, and C.L.McClendon (2007).
Reaching for high-hanging fruit in drug discovery at protein-protein interfaces.
  Nature, 450, 1001-1009.  
17568801 M.Kawasumi, and P.Nghiem (2007).
Chemical genetics: elucidating biological systems with small-molecule compounds.
  J Invest Dermatol, 127, 1577-1584.  
17961823 T.L.Gururaja, S.Yung, R.Ding, J.Huang, X.Zhou, J.McLaughlin, S.Daniel-Issakani, R.Singh, R.D.Cooper, D.G.Payan, E.S.Masuda, and T.Kinoshita (2007).
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  Chem Biol, 14, 1105-1118.  
16484997 A.Whitty, and G.Kumaravel (2006).
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17009316 D.C.Fry (2006).
Protein-protein interactions as targets for small molecule drug discovery.
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16902917 T.Berg (2006).
Inhibition of TNF-alpha signaling: divide and conquer.
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16945019 T.Chen, and X.Feng (2006).
Cell-based assay strategy for identification of motif-specific RANK signaling pathway inhibitors.
  Assay Drug Dev Technol, 4, 473-482.  
16408085 T.Clackson (2006).
Breaking and entering.
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16962311 V.Neduva, and R.B.Russell (2006).
Peptides mediating interaction networks: new leads at last.
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The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.