Structural basis of thalidomide enantiomer binding to cereblon

Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. However, the precise mechanism by which the two isomers exert their different activities remains poorly understood. Here, we present structural and biochemical studies of (S)- and (R)-enantiomers bound to the primary target of thalidomide, cereblon (CRBN). Our biochemical studies employed deuterium-substituted thalidomides to suppress optical isomer conversion, and established that the (S)-enantiomer exhibited ~10-fold stronger binding to CRBN and inhibition of self-ubiquitylation compared to the (R)-enantiomer. The crystal structures of the thalidomide-binding domain of CRBN bound to each enantiomer show that both enantiomers bind the tri-Trp pocket, although the bound form of the (S)-enantiomer exhibited a more relaxed glutarimide ring conformation. The (S)-enantiomer induced greater teratogenic effects on fins of zebrafish compared to the (R)-enantiomer. This study has established a mechanism by which thalidomide exerts its effects in a stereospecific manner at the atomic level.

(b) As in a, but for (R)-thalidomide (R-thal) before (top) and after (Bottom) the zebrafish experiments (see Supplementary Table 1).
(c) As in b, but for for deuterated (S)-Thalidomide, (S)-D-Thal, before (top) and after (Bottom) the auto-ubiquitylation experiments (see Supplementary Table 1). The pH of the medium of 6.54.
(d) As in c, but for deuterated (R)-Thalidomide, (R)-D-Thal. Before the experiment, the peak area is 45,608 μV/sec for R-D-Thal but no peak was detected for S-D-Thal. After the experiment, the peak area is 31,517 μV/sec for R-D-Thal but no peak was detected for S-D-Thal.
(e) HPLC elution profiles of racemic thalidomide for confirmation of the 1:1 enantiomer ratio. The peak areas are 337,651 μV/sec for S-Thal and 337,894 μV/sec for R-Thal, suggesting that our racemic thalidomide is a 1.0000:1.0007 racemate. (a) Binding of racemic thalidomide to TBD of human CRBN. Reverse titration was performed with injections of 1.5 μl of 1,500 μM TBD into 150 μM racemic thalidomide (250 μl) at 20°C. The injections were performed over a period of 4 sec with a 300-sec interval between injections, and the final concentration reached was 200 μM (26 injections total). TBD exhibited a K D value of 18.1±1.8 μM, with ΔH and TΔS values of -10.7 ± 0.3 and -4.3 kcal/mol, respectively. (b) As in a, but for binding of (S)-thalidomide (S-thal) to TBD of human CRBN. TBD exhibited a K D value of 3.5 ± 0.4 μM, with ΔH and TΔS values of -9.0 ± 0.2 and -1.6 kcal/mol, respectively. (c) As in a, but for binding of (R)-thalidomide (R-thal) to TBD of human CRBN. TBD exhibited a K D value of 20.0 ± 2.9 μM, with ΔH and TΔS values of -9.9 ± 0.5 and -3.6 kcal/mol, respectively. (d) As in a, but for binding of racemic thalidomide to TBD of mouse CRBN. TBD exhibited a K D value of 19.5 ± 2.2 μM, with ΔH and TΔS values of -12.0 ± 0.5 and -5.5 kcal/mol, respectively. (e) As in a, but for binding of (S)-thalidomide (S-thal) to TBD of mouse CRBN. TBD exhibited a K D value of 5.7 ± 0.5 μM, with ΔH and TΔS values of -10.2 ± 0.1 and -3.0 kcal/mol, respectively. (f) As in a, but for binding of (R)-thalidomide (R-thal) to TBD of mouse CRBN. TBD exhibited a K D value of 50.3 ± 11.1 μM, with ΔH and TΔS values of -18.5 ± 4.1 and +12.7 kcal/mol, respectively. (g) Summary of the obtained K D values.

Supplementary Figure 3 Sequence comparison of CRBN TBDs.
Sequence comparison of CRBN TBDs from different sources. The secondary structures of the free form of mouse CRBN TBD, which we previously reported 28 , are shown at the top with β-strands (β1-β8; blue arrows), 3 10 -helices (η1-η4; pink bars), loop (solid lines) and disordered residues (dotted lines). β-strands form anti-parallel β-sheets, the β1-β2-β8 strands and the β4-β5-β6-β7-β3 strands in (Figure 2 b). Three short 3 10 -helices: (η1,η3 and η4) are flanking the N-or C-terminal ends of the β-strands (β2, β3 and β7, respectively) and the last (η2) is located at the tip of the long flexible β2-β3 loop, which is disordered in the thalidomide-binding TBD (Figure 3 b and c). Acidic (D, E) and basic (K, R, H) residues are designated with red and blue letters, respectively. Residues which form the tri-Trp pocket and directly contact thalidomide are marked with cyan circles. Completely conserved residues are highlighted in blue and zinc-coordinated cysteines of the CXXC motifs in red. Residues involved in the nonsense mutation R419X of human CRBN are highlighted in orange. Mice and humans have only three amino acid differences: mouse Ser369 (loop β3-β4), Val380 (loop β4-β5) and Ile391 (β5) are replaced with Cys366, Glu377 and Val388 in humans, respectively.

Supplementary Figure 5
Structure of CRBN TBD prepared in the presence of racemic thalidomide.
(a) A stereoview of mouse CRBN TBD (cyan) bound to thalidomide (space-filling model; yellow for C, blue for N and red for O) prepared with racemic thalidomide. The glutarimide group of thalidomide is docked into the tri-Trp pocket (three Trp and single Val, His and Phe residues shown as stick models). The bound thalidomide was found to be the (S)-enantiomer. (b) A stereoview of the composite omit electron density map for thalidomide bound to tri-Trp pocket in mouse TBD. The map is shown as a red mesh in 1σ contour. The bound thalidomide is the (S)-enantiomer (yellow) displaying the C4-endo puckered conformation, as observed in the (S)-thalidomide-bound form prepared with (S)-thalidomide. The crystal structure was solved and refined with protein, ion and water molecules but without thalidomide molecules the map corresponding to the bound thalidomide molecules was calculated. The (S)-thalidomide (yellow) molecule, but not (R)-thalidomide, fits into the electron density. Summary of thalidomide racemization, main thalidomide metabolites and major hydrolysis pathways. Hydrolysis yields primary products α-(ocarboxybenzamido) glutarimide (I), phthaloylglutamine (II), or phthaloylisolglutamine (III) by ring opening. Compounds in pathway A retain the intact glutarimide ring, while compounds in pathway B retain the phthalimido ring but not the glutarimide ring, and compounds in pathway C have both rings opened.