Covalent Adducts Formed by the Androgen Receptor Transactivation Domain and Small Molecule Drugs Remain Disordered

Abstract

Intrinsically disordered proteins are implicated in many human diseases. Small molecules that target the disordered androgen receptor transactivation domain have entered human trials for the treatment of castration-resistant prostate cancer. These molecules have been shown to react with cysteine residues of the androgen receptor transactivation domain and form covalent adducts under physiological conditions. It is currently unclear how the covalent attachment of these molecules alters the conformational ensemble of the androgen receptor. Here, we utilize all-atom molecular dynamics computer simulations to simulate covalent adducts of small molecule ligands EPI-002 and EPI-7170 bound to the disordered androgen receptor transactivation domain. Our simulations reveal that the conformational ensembles of androgen receptor transactivation domain covalent adducts are heterogeneous and disordered. We find that covalent attachment of EPI-002 and EPI-7170 increases the population of collapsed helical transactivation domain conformations relative to the populations observed in non-covalent binding simulations, and we identify networks of protein-ligand interactions that stabilize collapsed conformations in covalent adduct ensembles. We compare the populations of protein-ligand interactions observed in covalent adduct ensembles to those observed in non-covalent ligand-bound ensembles and find substantial differences. Our results provide atomically detailed descriptions of covalent adducts formed by small molecules and an intrinsically disordered protein and suggest strategies for developing more potent covalent inhibitors of intrinsically disordered proteins.

1

Covalent attachment of EPI-002 and EPI-7170 stabilizes collapsed helical molten-globule-like states of the Tau-5 _{R2_R3} region of the androgen receptor transactivation domain. A) Chemical structures of EPI-002 and EPI-7170 and covalent adducts of EPI-002 and EPI-7170 attached to a cysteine residue. B) Helical propensities obtained from 300 K replicas of REST2 MD simulations. Helical propensities are shown for apo Tau-5_{R2_R3} (red), the Tau-5_{R2_R3}-CYS404:EPI-002 covalent adduct (green), the Tau-5_{R2_R3}-CYS404:EPI-7170 covalent adduct (purple), a non-covalent ligand-bound ensemble of Tau-5_{R2_R3} and EPI-002 (blue), and a non-covalent ligand-bound ensemble of Tau-5_{R2_R3} and EPI-7170 (orange). Simulated helical propensities are presented as mean values ± statistical error estimates from blocking. C) Free energy of Tau-5_{R2_R3} conformations in each ensemble as a function of the helical collective variable Sα. D) Free energy surfaces as a function of the radius of gyration (R _g ) and Sα. The dotted white lines indicate the defined boundary of the “helical globule” state (Sα > 6.0, R _g < 1.3 nm). The population of the helical globule state in each ensemble is reported as p _Glob .

2

t-SNE clustering of Tau-5 _{R2_R3} conformational states. t-SNE projections and cluster assignments of conformations from Tau-5_{R2_R3}-CYS404:EPI-002 and Tau-5_{R2_R3}-CYS404:EPI-7170 covalent adduct ensembles (A, B) and Tau-5_{R2_R3} conformations obtained from non-covalent EPI-002 and EPI-7170 ligand-binding simulations (C, D). Cluster assignments were obtained by performing t-SNE clustering on a merged ensemble containing the EPI-002 and EPI-7170 covalent adduct ensembles with N = 4 and N = 20 clusters and by performing t-SNE clustering on a merged ensemble containing all frames (bound and unbound) from EPI-002 and EPI-7170 non-covalent ligand-binding simulations with N = 4 and N = 18 clusters. t-SNE projections and average helical propensities of each t-SNE cluster are colored according to cluster assignments. Helical propensity is presented as mean values ± statistical error estimates from blocking.

3

Protein–ligand interactions in Tau-5 _{R2_R3} covalent adduct ensembles. A) t-SNE projections of covalent adduct ensembles of Tau-5_{R2_R3}-CYS404:EPI-002 and Tau-5_{R2_R3}-CYS404:EPI-7170 obtained with N = 4 clusters. Colored dots correspond to conformations in the merged ensemble of both covalent adducts, and black dots represent conformations from the specified individual covalent adduct ensemble. Illustrative snapshots of Tau-5_{R2_R3} are shown for selected subensembles of each cluster. A representative conformation of each subensemble is shown as a cartoon with the R2 and R3 regions of Tau-5_{R2_R3} colored red and blue, respectively, and the covalently attached ligand colored cyan. Backbone traces of additional conformations are shown as transparent tubes. B) Populations of intramolecular contacts and specific intramolecular interactions observed between covalently modified CYS404:EPI-002 and CYS404:EPI-7170 residues and Tau-5_{R2_R3} residues in each cluster.

4

Protein–ligand interactions in Tau-5 _{R2_R3} non-covalent ligand binding simulations of EPI-002 and EPI-7170. A) t-SNE projections of Tau-5_{R2_R3} conformations from non-covalent ligand binding simulations obtained with N = 4 clusters. Colored dots correspond to conformations in a merged ensemble from both binding simulations, and black dots represent conformations from the specified individual binding simulation. Illustrative snapshots of Tau-5_{R2_R3} are shown for selected subensembles of each cluster. A representative conformation of each subensemble is shown as a cartoon with the R2 and R3 regions of Tau-5_{R2_R3} colored red and blue, respectively. Backbone traces of additional conformations are shown as transparent tubes. The location of the bisphenol A scaffold of EPI ligands is shown for selected illustrative conformations in cyan. B) Populations of intermolecular contacts and specific intermolecular interactions observed between Tau-5_{R2_R3} and EPI-002 (blue) and Tau-5_{R2_R3} and EPI-7170 (orange) in each cluster.