Targeted modification of intracellular compounds

Process for the production of a targeted effector having the capacity to specifically modulate the properties of an intracellular target molecule T, and/or a cellular component C which interacts directly or indirectly in a cell with T, said process comprising the steps:

• a) production of a random pool of peptides, so-called recognition moieties R,
• b) screening of the random pool produced in (a) against T in a cell, in conditions suitable to allow identification of recognition moieties R capable of interacting with T,
• c) optionally contacting the moieties selected in (b) with proteins other than T to determine the specificity range of each of said moieties, and to identify moieties having a desired specificity range,
• d) covalent linkage of the recognition moieties R to an effector moiety E, E being a molecule which initially has the capacity to exert a predetermined effect on at least one intracellular component M, step (d) being carried out either prior to step (b), or after step (e),
• e) verification of the affinity A, with which the recognition moiety R interacts with T, or of the affinity A2 with which the targeted effector, interacts with T,
• f) if both of A1 and A2 correspond to Kd values greater than 1×10−8 M, alteration of the binding region of the recognitio moiety to adjust the binding affinity of the interaction between T and the selected moiety so that the Kd becomes less than 1×10−8 M.

Process for conferring on an effector moiety E the ability to specifically modulate the properies of an intracellular protein T, or an intracellular component C which interacts directly or indirectly with T,

• said process comprising the steps of
• i) covalently linking the effector moiety E to a recognition moiety R,
• wherein R comprises a molecule having the capacity to specifically interact, within a cell, with a site on an intracellular target molecule T, the interaction with T occurring with an affinity A1 which corresponds to a Kd value of less than 1×10−8 M, and
• E being a molecule which has an initial capacity to exert an effect on at least one molecule M, and which when it is covalently linked to R, acquires the capacity to exert the effect on the intracellular target molecule T,
• ii) optionally optimising the affinity of the interaction between T and R by altering the chemical composition of the binding region of R to provide an affinity in the desired range.

Dimeric intracellular recognition molecule, comprising two covalently linked intracellular recognition molecules R1 and R2,

• wherein R1 and R2 each comprise a recognition domain V, V being a peptide having a length of five to sixty amino acids, conformationally constrained by covalent bonding to a platform,
• R1 having the capacity to specifically interact, within a cell, with a site on a predetermined intracellular target molecule T1, and
• R2 having the capacity to specifically interact, within a cell, with a site on a predetermined intracellular target molecule T2,
• wherein R1 and R2 may be the same or different and T1 and T2 are distinct.

Process for the identification of dimeric recognition moieties having the capacity to force an interaction between two target molecule T1 and T2, and/or a cellular component C which interacts directly or indirectly in a cell with T1 or T2 said process comprising the steps

• a) production of a random pool of peptides, so-called recognition moieties R,
• b) screening of the random pool produced in (a) against T1 and T2 in a cell, in conditions suitable to allow identification of recognition moieties R capable of interacting with T1 or T2,
• c) optionally contacting the moieties selected in (b) with proteins other than T1 and T2 to determine the specificity range of each of said moieties, and to identify moieties having a desired specificity range,
• f) covalent linkage of two thus-identified recognition moieties R1 and R2 to each other to provide a dimeric recognition moiety,
• g) screening of the dimeric recognition moiety to identify moieties capable of interacting simultaneously with T1 and T2.