Identification and characterization of ternary complexes consisting of FKBP12, MAPRE1 and macrocyclic molecular glues

Abstract

Many disease-relevant and functionally well-validated targets are difficult to drug. Their poorly defined 3D structure without deep hydrophobic pockets makes the development of ligands with low molecular weight and high affinity almost impossible. For these targets, incorporation into a ternary complex may be a viable alternative to modulate and in most cases inhibit their function. Therefore, we are interested in methods to identify and characterize molecular glues. In a protein array screen of 50 different macrocyclic FKBP12 ligands against 2500 different randomly selected proteins, a molecular glue compound was found to recruit a dimeric protein called MAPRE1 to FKBP12 in a compound-dependent manner. The corresponding ternary complex was characterized by TR-FRET proximity assay and native MS spectroscopy. Insights into the 3D structure of the ternary complex were obtained by 2D protein NMR spectroscopy and finally by an X-ray structure, which revealed the ternary complex as a 2 : 2 : 2 FKBP12 : molecular glue : MAPRE1 complex exhibiting multiple interactions that occur exclusively in the ternary complex and lead to significant cooperativity α. Using the X-ray structure, rationally guided synthesis of a series of analogues led to the cooperativity driven improvement in the stability of the ternary complex. Furthermore, the ternary complex formation of the series was confirmed by cellular NanoBiT assays, whose A _max values correlate with those from the TR-FRET proximity assay. Furthermore, NanoBiT experiments showed the functional impact (inhibition) of these molecular glues on the interaction of MAPRE1 with its intracellular native partners.

Fig. 1. Simplification of the FKBP12 binding motif. Rapamycin and FK506 have the same 9 stereocenter-containing moiety (shown in red, left side), which has been structurally and synthetically simplified to a moiety with only 2 stereocenters, retaining most of the affinity for FKBP12. Changing the stereoconfiguration of the 4-ethyl-1,2-dimethoxybenzene substituent from R to S completely abolishes the binding affinity to FKBP12, giving an impression of the high specificity of even the simplified binding motif (right side).

Fig. 2. Supplementation of the FKBP12 binding motif by a diverse recruiting loop. While the binding of FKBP12 is very sensitive to structural changes in the binding moiety itself, this is not the case for its macrocyclic complementation. Peptidic loops with different numbers of residues, N-methylated or not, are tolerated, maintaining a low nM affinity to FKBP12.

Fig. 3. Macrocyclic scaffolds for FKBP12 ligands with divers and modular recruitment loops. (left) Dipeptidic recruitment loop using Lys side-chain cyclization leads to an “exocyclic” amino group as a diversity vector for functionalization as amides, ureas or amines. (right) The di- or tripeptidic recruitment loop with head-to-tail cyclization leads to an “endocyclic” diversity originating exclusively from the amino acid residues.

Fig. 4. Schematic representation of the protein array screening. On a protein array glass surface, approximately 2500 randomly collected proteins were incubated with fluorescently labelled FKBP12 (FL-FKBP12). After washing the surface, no novel residual fluorescence was observed on the surface in the absence of any of the 50 selected macrocyclic FKBP12 ligands. In contrast, in the presence of one of the 50 compounds per run, one compound caused novel residual fluorescence on the surface at one position (n = 2), indicating compound-dependent ternary complex formation.

Fig. 5. MAPRE1 is the only one out of 2500 proteins recruited by 1 out of 50 FKBP12 tested ligands into a ternary complex MAPRE1:R,S-SLF-1a:FKBP12. Model of full length MAPRE1 structure based on 2QJZ (N-term domain) and 1TXQ (C-term domain). MAPRE1 belongs to the family of end-binding proteins (EB) and is a “bifunctional” protein that binds to microtubules (MTs) and brings them into proximity to plus end tracking proteins (+TIPs).

Fig. 6. TR-FRET proximity assay that validates the ternary complex formation by the screening sample and the resynthesized single isomer involving the coiled coil domain of MAPRE1. The screening sample SLF-1 (diastereomeric mixture) brings full-length MAPRE1 (red) and its coiled-coil-containing N-terminal domain (green) into proximity with FKBP12 in a compound-dependent manner, whereas the C-terminal domain of MAPRE1 (yellow) does not form a ternary complex with SLF-1 and FKBP12 (upper part). Resynthesized R,S-SLF-1a confirms exclusive ternary complex formation with the C-terminus of MAPRE1, even with a higher A_max and assigns ternary complex formation to a single epimer (lower part).

Fig. 7. Assignment of absolute stereochemistry of separated building block enantiomers. The chiral separation of racemic Boc-4-methylenepiperidine-2-carboxylic acid led to two enantiomerically pure amino acids with undetermined absolute stereochemistry. Coupling each of them with l-Ala-OtBu and subsequent cyclization to the two diastereomeric diketopiperazines allowed the assignment of the absolute stereochemistry of the two 4-methylenepiperidine-2-carboxylic acids used. The enantiomer with the earlier retention time (1.42 min) led to the diketopiperazine with two α protons cis to each other, the enantiomer with the later retention time (2.09 min) to the diketopiperazine with the two α protons trans to each other.

Fig. 8. Relation between enantiomerically pure 4-methylenepiperidine-2-carboxylic acid and corresponding macrocyclic FKBP12 ligands. Enantiomerically pure and assigned (R)- and (S)-Boc-4-methylenepiperidine-2-carboxylic acid enabled the synthesis of the single isomers R,S-SLF-1a (left) and S,S-SLF-1d (right).

Fig. 9. Assignment of recruitment activity to the epimer R,S-SLF-1a. Only the single isomer R,S-SLF-1a confirmed the initially observed activity, while S,S-SLF-1d was completely inactive, indicating a high specificity of recruitment of MAPRE1 to the preformed binary R,S-SLF-1a:FKBP12 complex.

Fig. 10. Native MS of the ternary complex FKBP12 : R,S-SLF-1a : MAPRE1 (2 : 2 : 2). Conditions: 10 μM FKBP and 10 μM MAPRE1 with 25 μM ligand, 1% DMSO. Top panel: FKBP1a with MAPRE1. The primary species observed of MAPRE1 were both monomer (single green diamond) and dimer (double green diamond). FKBP12 monomer was the main species observed for FKBP12 (partial blue circle). Bottom panel: Ternary complex formation mediated by R,S-SLF-1a. The species observed were as follows: free FKBP12 (partial blue circle), FKBP12 + R,S-SLF-1a (partial blue circle with small yellow circle) and MAPRE1 was mainly observed as dimer (double green diamond). Additionally, a ternary complex composed of 1× FKBP12 + 1× R,S-SLF-1a + MAPRE1 dimer (partial blue circle with small yellow circle and double green diamond) and a complex composed of 2× FKBP1a + 2× R,S-SLF-1a + MAPRE1 dimer (2× partial blue circle with small yellow circle and double green diamond).

Fig. 11. NMR characterization of the ternary complex FKBP12 : R,S-SLF-1a : MAPRE1 (2 : 2 : 2). (A) ¹⁵N-HSQC NMR spectrum of ¹³C,¹⁵N-MAPRE1 (100 μM, black) is superimposed on a ¹⁵N-HSQC NMR spectrum of ¹³C,¹⁵N-MAPRE1 in presence of FKBP12:R,S-SLF-1a, generating the 2 : 2 : 2 ternary complex. Resonance assignments are given in the figure. (B) MAPRE1 residues that are affected by the addition of FKBP12:R,S-SLF-1a are mapped onto the X-ray structure of the ternary complex. FKBP12 is shown in light cyan, R,S-SLF-1a is shown in blue, and MAPRE1 is shown in gray, except for the residues affected in the ternary complex, which are colored dark red or light red, according to the magnitude of the effect.

Fig. 12. NMR investigation of weak intrinsic interactions in binary complexes. (A) The ¹⁵N-HSQC NMR spectrum of ¹³C,¹⁵N-MAPRE1 (50 μM, black) is superimposed on a ¹⁵N-HSQC NMR spectrum of ¹³C,¹⁵N-MAPRE1 in presence of 100 μM R,S-SLF-1a, Note that the solubility of R,S-SLF-1a is only about 20 μM. Resonance assignments of the two residues that experience chemical shift changes are given in the figure. (B) The ¹⁵N-HSQC NMR spectrum of ¹³C,¹⁵N-MAPRE1 (50 μM, black) is superimposed on a ¹⁵N-HSQC NMR spectrum of ¹³C,¹⁵N-MAPRE1 in presence of 350 μM FKBP12, There are no chemical shift changes in the presence of FKBP12, indicating no intrinsic affinity between the two proteins in the absence of R,S-SLF-1a.

Fig. 13. X-ray structure of the ternary complex FKBP12 : R,S-SLF-1a : MAPRE1 (2 : 2 : 2). R,S-SLF-1a acts as a molecular glue with a predominant hydrophobic interface bridging MAPRE1 and FKBP12. Limited protein–protein interactions flank the R,S-SLF-1a binding pocket. PDB code: 9CO5/DOI: https://doi.org/10.2210/pdb9co5/pdb.

Fig. 14. X-ray structure of the binary complex FKBP12:R,S-SLF-1a (green and cyan). Purple: R,S-SLF-1a in ternary complex. A conformational change of the solvent exposed loop of R,S-SLF-1a is observed upon MAPRE1 recruitment.

Fig. 15. Structural modifications around the original hit compound R,S-SLF-1a.

Fig. 16. Cellular recruitment of MAPRE1 to FKBP12 by R,S-SLF-1a analogs correlates with biochemical results. (A) Schematic of the cellular NanoBiT assay. (B) NanoBiT dose response curves with R,S-SLF-1a and analogues. (C) Scatter plot of TR-FRET recruitment A_maxversus NanoBiT recruitment A_max. (D) Dependency on FKBP12 ligand binding site for MAPRE1 recruitment. Dose response of the 12 SLF analogs with the highest %A_max values from panel (A) but in the presence of 80 μM of the indicated competitive non-recruiting FKBP12 ligand or DMSO.

Fig. 17. Recruitment of MAPRE1 to FKBP12 by R,S-SLF-1a is disrupted by forced MAPRE1-CEP215 interaction in cells. (A) Schematic of NanoBiT competition experiment. LgBiT-FKBP12 and SmBiT-MAPRE1 create a signal only in the presence of R,S-SLF-1a, which can be competed by co-expressing untagged CEP215 binding to MAPRE1. (B) Overexpressed WT CEP215, but not mutant CEP215, strongly diminishes recruitment of MAPRE1 to FKBP12 (R,S-SLF-1a = strong recruiter; Cpd 4 = R,S-SLF-1d = strong ligand of FKBP12 but non-recruiting epimer of R,S-SLF-1a).