procedure as with a standard amino acid. Biotin will be soluble
in DMF once DIPEA has been added to the solution.
17. Addition of a negative control, for example an inactive alanine
mutant.
18. For one mouse brain (m ¼ 0.4 g), use 4 mL of homogenization
buffer and do not move grinder above solvent level
19. As a negative control for the primary antibodies use protein
lysates from hippocampal tissue of previously described mutant
mice bearing a loss-of-function mutation in the Dlg1
(SAP-97), Dlg2 (PSD-93), Dlg3 (SAP-102), and Dlg4
(PSD-95) genes.
20. If samples contain serum or phenol red it can affect the readout intensity. Use serum or phenol free media if possible or use
a background control
21. Procaine (positive control) and procainamide (negative control) were investigated at 50 μM.
22. We recommend to store the MTT reagent at 4
C in the dark as
it is sensitive to light.
Acknowledgments
The chapter was supported by EU Horizon 2020 RIA under the
Marie Skłodowska-Curie grant agreement no. 675341
References
1. Wells JA, McClendon CL (2007) Reaching for
high-hanging fruit in drug discovery at protein–protein
interfaces.
Nature
450:1001–1009
2. De Las Rivas J, Fontanillo C (2010) Protein–protein interactions essentials: key concepts to
building and analyzing interactome networks.
PLoS Comput Biol 6:e1000807
3. Scott DE, Bayly AR, Abell C, Skidmore J
(2016) Small molecules, big targets: drug discovery faces the protein–protein interaction
challenge. Nat Rev Drug Discov 15:533–550
4. Sundell
GN,
Arnold
R,
Ali
M,
Naksukpaiboon P, Orts J, Gu ¨ntert P, Chi CN,
Ivarsson Y (2018) Proteome-wide analysis of
phospho-regulated PDZ domain interactions.
Mol Syst Biol 14:e8129
5. Fanning AS, Anderson JM (1996) Protein–protein interactions: PDZ domain networks.
Curr Biol 6:1385–1388
6. Yu Y, Li S, Wang K, Wan X (2019) A PDZ
protein MDA-9/Syntenin: as a target for
cancer therapy. Comput Struct Biotechnol J
17:136–141
7. Zeng M, Shang Y, Guo T, He Q, Yung W-H,
Liu K, Zhang M (2016) A binding site outside
the canonical PDZ domain determines the specific interaction between Shank and SAPAP and
their function. Proc Natl Acad Sci U S A 113:
E3081–E3090
8. Mu ¨nz M, Hein J, Biggin PC (2012) The role
of flexibility and conformational selection in
the binding promiscuity of PDZ domains.
PLoS Comput Biol 8:e1002749
9. Schreiber G, Keating AE (2011) Protein binding specificity versus promiscuity. Curr Opin
Struct Biol 21:50–61
10. Hill MD, Goyal M, Menon BK, Nogueira RG,
McTaggart RA, Demchuk AM et al (2020)
Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPENA1): a multicentre, double-blind, randomised controlled trial. Lancet 395:878–887
176
Dominik J. Essig et al.
in DMF once DIPEA has been added to the solution.
17. Addition of a negative control, for example an inactive alanine
mutant.
18. For one mouse brain (m ¼ 0.4 g), use 4 mL of homogenization
buffer and do not move grinder above solvent level
19. As a negative control for the primary antibodies use protein
lysates from hippocampal tissue of previously described mutant
mice bearing a loss-of-function mutation in the Dlg1
(SAP-97), Dlg2 (PSD-93), Dlg3 (SAP-102), and Dlg4
(PSD-95) genes.
20. If samples contain serum or phenol red it can affect the readout intensity. Use serum or phenol free media if possible or use
a background control
21. Procaine (positive control) and procainamide (negative control) were investigated at 50 μM.
22. We recommend to store the MTT reagent at 4
C in the dark as
it is sensitive to light.
Acknowledgments
The chapter was supported by EU Horizon 2020 RIA under the
Marie Skłodowska-Curie grant agreement no. 675341
References
1. Wells JA, McClendon CL (2007) Reaching for
high-hanging fruit in drug discovery at protein–protein
interfaces.
Nature
450:1001–1009
2. De Las Rivas J, Fontanillo C (2010) Protein–protein interactions essentials: key concepts to
building and analyzing interactome networks.
PLoS Comput Biol 6:e1000807
3. Scott DE, Bayly AR, Abell C, Skidmore J
(2016) Small molecules, big targets: drug discovery faces the protein–protein interaction
challenge. Nat Rev Drug Discov 15:533–550
4. Sundell
GN,
Arnold
R,
Ali
M,
Naksukpaiboon P, Orts J, Gu ¨ntert P, Chi CN,
Ivarsson Y (2018) Proteome-wide analysis of
phospho-regulated PDZ domain interactions.
Mol Syst Biol 14:e8129
5. Fanning AS, Anderson JM (1996) Protein–protein interactions: PDZ domain networks.
Curr Biol 6:1385–1388
6. Yu Y, Li S, Wang K, Wan X (2019) A PDZ
protein MDA-9/Syntenin: as a target for
cancer therapy. Comput Struct Biotechnol J
17:136–141
7. Zeng M, Shang Y, Guo T, He Q, Yung W-H,
Liu K, Zhang M (2016) A binding site outside
the canonical PDZ domain determines the specific interaction between Shank and SAPAP and
their function. Proc Natl Acad Sci U S A 113:
E3081–E3090
8. Mu ¨nz M, Hein J, Biggin PC (2012) The role
of flexibility and conformational selection in
the binding promiscuity of PDZ domains.
PLoS Comput Biol 8:e1002749
9. Schreiber G, Keating AE (2011) Protein binding specificity versus promiscuity. Curr Opin
Struct Biol 21:50–61
10. Hill MD, Goyal M, Menon BK, Nogueira RG,
McTaggart RA, Demchuk AM et al (2020)
Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke (ESCAPENA1): a multicentre, double-blind, randomised controlled trial. Lancet 395:878–887
176
Dominik J. Essig et al.