In vivo engineered cereblon protein
Inventors
Weinstein, David • Cravatt, Benjamin F. • Patricelli, Matthew • Stamos, Dean • SIMON, Gabe • HORNING, Benjamin • VINOGRADOVA, EKATERINA • Nordin, Brian • BALTGALVIS, Kristen • Kinsella, Todd
Assignees
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Publication Number
US-10781239-B2
Publication Date
2020-09-22
Expiration Date
Abstract
Disclosed herein are in vivo engineered cereblon protein and methods of making the same. The in vivo engineered cereblon protein can include a site-specific non-naturally occurring modification at cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO:2 or 3, the modification comprising a moiety resulting from an in vivo Michael addition reaction between an exogenous Michael acceptor and the cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO:2 or 3.
Core Innovation
The invention relates to an in vivo engineered cereblon protein that includes a non-naturally occurring covalent modification at cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO:2 or 3. The covalent modification results from an in vivo Michael addition reaction between an exogenous Michael acceptor and the cysteine residue, in which a sulfur atom at the cysteine residue undergoes the Michael reaction with a double bond of the exogenous Michael acceptor.
The engineered cereblon is characterized by reduced engagement potency at cysteine 318 as set forth in SEQ ID NO:1 or at cysteine 317 as set forth in SEQ ID NO:2 or 3. The document describes that IMiDs such as pomalidomide and CC-220 bind cereblon and inhibit labeling of cereblon cysteines C287 and C318, and that IMiD binding causes conformational changes that block an engineered Michael-addition/labeling pocket.
The document further provides chemical probes and examples, including 4-phenoxyazetidine cyanoacrylamides and related cereblon compounds, and describes examples for installing electrophiles or Michael acceptor motifs onto piperidine-oxy substituted heteroaryl scaffolds. It also states that modification at C287 can inhibit PROTAC dFKBP13 function and describes assessment of global promiscuity by probe-label inhibition across targets.
Claims Coverage
The provided material identifies one independent claim covering an in vivo engineered cereblon protein with a non-naturally occurring covalent Michael addition modification at a specified cysteine, together with reduced engagement potency at an alternate cysteine site. Dependent claims further refine the covalent modifier selection, modification specificity, engagement behavior, and charged interaction residues.
In vivo engineered covalent Michael modification at specified cereblon cysteine
An in vivo engineered cereblon protein comprising a non-naturally occurring covalent modification at cysteine 287 as set forth in SEQ ID NO:1, or cysteine 286 as set forth in SEQ ID NO:2 or 3, where the modification results from an in vivo Michael addition reaction between an exogenous Michael acceptor and the specified cysteine.
Sulfur atom covalently undergoes Michael reaction with acceptor double bond
The engineered cereblon modification is defined such that a sulfur atom at the cysteine residue undergoes the Michael reaction with a double bond of the exogenous Michael acceptor.
Reduced engagement potency at alternate cereblon cysteine site
The engineered cereblon has reduced engagement potency at cysteine 318 as set forth in SEQ ID NO:1 or at cysteine 317 as set forth in SEQ ID NO:2 or 3.
Exogenous Michael acceptor promiscuity constraint
The exogenous Michael acceptor has global promiscuity no greater than 10% at 500 µM.
Exogenous Michael acceptor TE50 threshold
The exogenous Michael acceptor has a TE50 of no greater than 80 µM.
Specific exogenous Michael acceptor: acrylamide
The exogenous Michael acceptor is acrylamide.
Modification restricted to a single cysteine position
The engineered cereblon is only modified at cysteine 287 or only at cysteine 286.
Charged-residue engagement refinement
The exogenous Michael acceptor forms a charged interaction with one or more specified residues selected from Asp279, Asp 265, Arg 162, Arg 283, Lys 324, and Arg 419.
Overall, the claim coverage centers on an in vivo engineered cereblon containing a non-naturally occurring covalent Michael addition modification at Cys287 or Cys286 via an exogenous Michael acceptor, with the sulfur undergoing reaction to the acceptor’s double bond, and with reduced engagement potency at the alternate Cys318 or Cys317 site.
Stated Advantages
Reduced engagement potency at cysteine 318 or cysteine 317.
Prevention of IMiD-pocket-dependent binding.
Reduced or abolished engagement of the alternate cysteine site.
Inhibition of cereblon-dependent PROTAC degradation.
Inhibition of IMiD binding.
Documented Applications
Inhibition of cereblon-dependent PROTAC degradation.
Modulation or inhibition of IMiD binding.
Inhibition of labeling of cereblon cysteines C287 and C318 by IMiDs such as pomalidomide and CC-220 using chemical probes.
Assessment of global promiscuity by probe-label inhibition across targets.
Context that C287 modification can inhibit PROTAC dFKBP13 function.
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