US 11,740,244 B2
Methods of screening for modulators of binding between GTPase and GTPase binding domains
Stéphanie Cabantous, Toulouse (FR); Faten Koraïchi, Toulouse (FR); and Gilles Favre, Toulouse (FR)
Assigned to INSERM (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE), Paris (FR); and UNIVERSITE PAUL SABATIER TOULOUSE III, Toulouse (FR)
Filed by INSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE), Paris (FR); and UNIVERSITÉ PAUL SABATIER TOULOUSE III, Toulouse (FR)
Filed on Aug. 6, 2020, as Appl. No. 16/986,551.
Application 16/986,551 is a division of application No. 15/736,200, granted, now 10,782,304, previously published as PCT/EP2016/064612, filed on Jun. 23, 2016.
Claims priority of application No. 15305971 (EP), filed on Jun. 24, 2015; and application No. 15306542 (EP), filed on Sep. 30, 2015.
Prior Publication US 2021/0041450 A1, Feb. 11, 2021
Int. Cl. G01N 33/68 (2006.01); G01N 33/542 (2006.01); C07K 16/18 (2006.01); C40B 30/04 (2006.01); G01N 33/50 (2006.01); G01N 33/533 (2006.01)
CPC G01N 33/6845 (2013.01) [C07K 16/18 (2013.01); C40B 30/04 (2013.01); G01N 33/5008 (2013.01); G01N 33/533 (2013.01); G01N 33/542 (2013.01); C07K 2319/70 (2013.01)] 2 Claims
 
1. A method for screening a compound capable of modulating the binding between a polypeptide (A) and a polypeptide (B) comprising the step of
i) providing a cell that expresses:
(a) a polypeptide (GFP1-9) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence selected from the group consisting of SEQ ID NO:1-4
(b) a first fusion protein wherein the polypeptide (A) is fused to a polypeptide (GFP10) having an amino an amino acid sequence having at least 90% of identity with the amino acid sequence selected from the group consisting of SEQ ID NO:5-7
(c) a second fusion protein wherein the polypeptide (B) is fused to a polypeptide (GFP11) having an amino an amino acid sequence having at least 90% of identity with the amino acid sequence selected from the group consisting of SEQ ID NO:8-9
and (d) an intrabody specific for the complex formed by the self-assembly of the first, second and third polypeptides (a), (b) and (c), wherein the intrabody is a single domain antibody comprising the three complementarity determining regions (CDRs) of SEQ ID NO: 16;
ii) contacting the cell with a candidate compound,
iii) detecting the fluorescence; and
iv) positively selecting the candidate compound when the fluorescence is modulated compared to fluorescence detected in the absence of the candidate compound,
wherein the polypeptide (A) is a GTPase and the polypeptide (B) is a GTPase binding domain (GBD) or vice versa.