Molecular Medicine Israel

Activation of parkin by a molecular glue

Abstract

Mutations in parkin and PINK1 cause early-onset Parkinson’s disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson’s disease.

Introduction

Mitochondrial homeostasis is a critical process for neuronal survival. Mutations in several mitochondrial proteins that regulate the dynamics and turnover of this organelle lead to specific neurodegenerative diseases1. In particular, mutations in parkin or PINK1 cause early-onset Parkinson’s disease (EOPD), a movement disorder arising from the loss of specific neurons including (but not exclusively) dopaminergic neurons in the midbrain that are responsible for fine motor control2,3. Parkin and PINK1 work together to mediate selective turnover of damaged mitochondria via macroautophagy (a process called mitophagy) or through formation of mitochondria-derived vesicles that deliver damaged cargo to lysosomes for degradation4. Defects in these pathways lead to mitochondria-linked inflammation and autoimmune responses that lead to neuronal death5,6,7,8. Furthermore, loss of soluble parkin in the human brain correlates with age9,10 and impaired mitophagy is associated with sporadic PD11. While the details of the pathways leading to cell loss remain to be elucidated, it is clear that parkin and PINK1 suppress these deleterious responses by targeting mitochondrial compartments for degradation.

The molecular mechanisms of parkin/PINK1 have been elucidated in detail over the last two decades12. Parkin is a member of the RING-In-Between-RING (RBR) family of E3 ubiquitin ligases, which carry a RING1 domain that binds E2 ubiquitin-conjugating enzymes, as well as a catalytic Rcat (or RING2) domain that transfers ubiquitin to a substrate via a thioester intermediate13. Parkin also has two zinc-finger domains called RING0 and IBR, as well as an N-terminal ubiquitin-like (Ubl) domain with a regulatory phosphorylation site (Fig. 1a)14,15. In the basal state, parkin adopts a conformation with three features that maintain auto-inhibition: (1) the Rcat domain binds to RING0, which blocks access to the active site Cys431 in Rcat, (2) the Repressor Element of Parkin (REP), located upstream of Rcat, binds to RING1 and occludes the E2-binding site, (3) the Ubl binds to RING1, which prevents Ubl phosphorylation by PINK1 at Ser6516,17,18,19. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane (OMM), where its cytosolic kinase domain phosphorylates Ser65 in ubiquitin20,21,22,23,24,25. Phospho-ubiquitin (pUb) recruits parkin to the OMM through a high-affinity site on the RING1 domain, which induces the release and phosphorylation of the Ubl domain19,26,27,28. In turn, the phosphorylated Ubl domain (pUbl) binds to RING0 and displaces the Rcat domain and REP, allowing the formation of the active thioester transfer complex with a charged E2~Ub conjugate29,30,31. This complex is further stabilized by the activation element (ACT) of parkin, located in the Ubl-RING0 linker, which makes contacts with both the pUbl and RING0 domains30. Active parkin then ubiquitinates OMM proteins such as Mfn2 and Miro, which leads to selective mitochondrial turnover32,33,34.

Pathogenic missense EOPD mutations can be found throughout parkin domains. Those have been categorized into 5 functional groups based on their normalized abundance and impact on mitophagy35. Groups 1 and 2 are clearly or likely pathogenic and have the greatest impact on mitophagy. Group 1 mutants affect protein folding and stability and include mutations in zinc-coordinating cysteines, as well as R275W in the RING1 domain. Group 2 mutants reduce mitophagy without affecting protein stability. Those include mutations in the active site (e.g., C431F), as well as mutations that prevent pUb or pUbl binding (e.g., K211N). Mutations that affect the PINK1-mediated activation such as Ubl mutations R42P and V56E, or K211N in RING0 which impairs pUbl-binding, could all be rescued by synthetic activating mutations that disrupt auto-inhibitory interactions. Those activating mutations include F146A in RING0 (Rcat and ACT interface), and W403A in the REP (RING1 interface), which can also rescue the parkin S65A mutation or Ubl deletion19,36. In a recent survey of synthetic activation mutants, we found that mutations that robustly increase parkin’s activity and can rescue the S65A mutation form a cluster at the junction of the REP-RING1 and RING0-Rcat interfaces, suggesting this is a hotspot for parkin activation37.

Activation of the parkin/PINK1 pathway is an attractive therapeutic strategy for the treatment of EOPD as well as sporadic PD38. Building on the discovery of kinetin as a PINK1 activator39, Mitokinin (acquired by Abbvie in 2023) developed potent small-molecules that stimulate PINK1-dependent mitophagy in the presence of mitochondrial damage (EC50 ~ 500 nM) and promote the clearance of α-synuclein aggregates, which are a hallmark of sporadic PD40. Inhibitors of USP30, a deubiquitinating enzyme on the OMM that counters parkin’s activity41, are also being developed by several companies and are currently under clinical evaluation. Other groups used phenotypic screens or computational approaches to identify small molecules that stimulate parkin/PINK1-mediated mitophagy, but the targets of these molecules remain unknown42,43. Parkin-activating compounds have also been pursued by several groups, with three patents reporting small-molecules that activate parkin-mediated mitophagy44,45,46. However, it is unclear how these compounds increase parkin activity, and whether they mediate their effect through parkin directly47. In 2022, our group at Biogen reported a class of small-molecule allosteric modulators that increase parkin’s E3 ubiquitin ligase activity in biochemical assays48. These tetrahydropyrazolo-pyrazine (THPP) compounds activate parkin in a pUb-dependent manner, demonstrate stereospecificity, and have submicromolar EC50 values. However, in the absence of structural data, the mechanism of action of these compounds remained unknown.

Here, we report the structural basis for the activation of parkin by THPP compounds. Using autoubiquitination assays and isothermal calorimetry (ITC), we show that binding of the most potent THPP compound BIO-2007817 (EC50 = 150 nM) is dependent on the binding of pUb or exogenous pUbl to RING0. Indeed, we recently discovered that pUb can directly activate parkin through binding the RING0 domain49,50. We built on this to determine the crystal structure of a related THPP compound BIO-1975900 bound to the complex of parkin RING0-RING1-IBR domains (R0RB) and two pUb molecules. The structure, confirmed by nuclear magnetic resonance (NMR) and mutagenesis, reveals that the THPP compounds bind to the RING0 domain and make contacts with the pUb molecule in the RING0 site (pUbl binding site), mimicking the ACT element. Structure-activity relationship (SAR) analysis of two subseries of THPP compounds establishes the importance of binding pockets in RING0 (near W183 and P180) and pUb (near K48). Based on this structural model, we hypothesized that the THPP compounds would rescue Ubl mutations. Indeed, we demonstrate that BIO-2007817 can partially rescue EOPD mutations R42P, V56E, as well S65A and ΔUbl using in organello ubiquitination assays and mitoKeima-based cellular mitophagy assays. These results show that RBR ligases can be modulated pharmacologically and highlight the clinical potential of THPP compounds in the treatment of EOPD.

Results

Ubl is not essential for parkin activation by BIO-2007817

Previous experiments showed that BIO-2007817 could trigger autoubiquitination activity of unphosphorylated parkin in the presence of pUb48. We confirmed those results with both human and rat parkin, which share 86% identity (Fig. 1b). At 200 µM, BIO-2007817 induced activity similar to phosphorylated (activated) parkin but the compound did not further stimulate the activity of phosphorylated parkin. As previously reported48, the activation was strictly dependent on the presence of pUb. To avoid charging of the E2 enzyme with pUb, a C-terminal deletion (pUbΔG76) was used. Activation could also be detected in ubiquitin-vinyl sulfone (Ub-VS) assays51 that measure the accessibility of Rcat and are not dependent on the upstream components of the ubiquitination cascade (Fig. 1d). We next asked if the parkin Ubl domain was required for activation. We observed equal autoubiquitination activity of full-length and R0RBR parkin (lacking the Ubl domain) in both the human and rat proteins (Fig. 1b). Thus, the Ubl domain is not implicated in the mechanism of activation.

BIO-2007817 requires pUb bound to RING0 domain

Phospho-ubiquitin can bind two distinct sites in parkin. The high-affinity site on RING1 mediates the recruitment of parkin to the mitochondria19. With an affinity of around 20 nM, it is formed by residues H302 and R305 and promotes detachment and phosphorylation of the Ubl domain52. The second site is located on the RING0 domain and regulates parkin activity. While the second site typically binds pUbl (when parkin is phosphorylated), we previously demonstrated that it can also bind pUb49. The site is formed by residues K211, R163 and K161 and with an affinity of around 400 nM for pUb and 1 µM for pUbl49,50. This binding forces the dissociation of Rcat domain and the release of parkin ligase activity.

We investigated which of the two sites is needed to promote activation by comparing the effects of a RING1 double mutation (H302A A320R) and a RING0 mutation (K211N). In the absence of compound, none of the parkin variants—wild-type, H302A A320R, or K211N—can autoubiquitinate in the presence of pUb (Fig. 1c). Upon addition of BIO-2007817, the double mutant H302A A320R was activated to close to wild-type levels as judged by the disappearance of unmodified parkin and appearance of a high molecular weight smear of autoubiquitinated parkin. In contrast, the K211N parkin mutant was strongly impaired with an absence of high molecular weight parkin adducts (Fig. 1c). Thus, pUb binding to RING0 but not RING1 is required for activation by BIO-2007817. We confirmed this result in the Ub-VS assay that measures Rcat release. At 2.5 µM BIO-2007817, half of wild-type parkin was modified by the Ub-VS reagent while the K211N mutation completely prevented parkin modification even at 200 µM (Fig. 1d). Taken together, these results demonstrate that activation by BIO-2007817 requires the binding of an exogenous pUb molecule to the RING0 binding site of parkin.

BIO-2007817 acts a molecular glue for RING0 and pUb/pUbl

We measured the affinity of BIO-2007817 for parkin using isothermal titration calorimetry (ITC) experiments with a parkin construct R0RB that lacks both the Ubl domain and the Rcat domain (Fig. 2, Supplementary Table 1). Since pUb and Rcat compete for binding to RING0, deleting the Rcat domain favors pUb binding. The initial experiment measured BIO-2007817 binding to R0RB with pUb bound to both the RING1 and RING0 sites (R0RB + two pUb). BIO-2007817 bound the complex with 10 nM affinity and one-to-one stoichiometry (Fig. 2a). The interaction was highly specific as the inactive diastereomer BIO-200781848 (Fig. 1e) bound with 150-fold less affinity (Fig. 2a).

We next asked if pUb was required for BIO-2007817 binding (Fig. 2b). In the absence of pUb, the affinity was decreased over 5000-fold (to the limit of detection), mirroring the requirement for pUb observed in autoubiquitination experiments. Loss of the RING0 pUb-binding site (due to the K211N mutation) similarly prevented binding (Fig. 2c). Although BIO-2007817 had no effect on phosphorylated parkin, surprisingly, the phosphorylated Ubl domain (pUbl) added in trans could replace pUb. In ITC experiments, BIO-2007817 bound to the R0RB:pUbl complex with the same affinity as the pUb complex (Fig. 2d). As the R0RB fragment does not undergo any conformational rearrangements and pUbl binds only to RING0, this suggests that BIO-2007817 binds at the RING0/pUb (or RING0/pUbl) interface.

Additional insight into the site of BIO-2007817 binding came from the observation that inclusion of the ACT region with the pUbl domain (pUbl-ACT) decreased the affinity of BIO-2007817 binding 10-fold (Fig. 2e). In contrast, in the absence of compound, the ACT improves the affinity of pUbl binding to R0BR three-fold50. This suggests they compete for a common binding site. Identification of the binding site was confirmed by a mutation in ACT-binding site. Addition of a single oxygen atom (F146Y) decreased BIO-2007817 binding 10-fold (Fig. 2f). As F146 is surface-exposed and does not make contacts with pUb or pUbl in crystal structures of parkin, this strongly suggests that F146 directly binds BIO-2007817.

To directly test if BIO-2007817 behaves as a molecular glue, we measured the affinity of the pUbl domain binding to RING0 in the R0RB construct. As previously reported49,50, pUbl in trans has low micromolar affinity (Fig. 2g). This contrasts with the situation in intact phosphorylated parkin when the pUbl domain is present in cis. Inclusion of a saturating concentration (200 µM) of BIO-2007817 increased the pUbl binding affinity by more than three orders of magnitude, demonstrating that the compound acts as a molecular glue (Fig. 2h).

Mechanism of activation by THPP compounds

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) has been instrumental for characterizing the conformational changes during parkin activation29,30,31. HDX-MS measures solvent exposure which provides information on protein structure and interdomain contacts. We performed a series of HDX-MS experiments to resolve the mechanism of parkin activation by THPP compounds (Fig. 3). As previously observed, addition of pUb to parkin increased deuteration of the Ubl domain (Fig. 3a). This reflects the release of the Ubl from its binding site on RING1, which facilitates its phosphorylation by PINK129,30,31. There was no increase in deuteration of Rcat domain as pUb binding to RING1 is not sufficient to activate parkin. In contrast, addition of BIO-2007817 in the presence of pUb, increased deuteration of the catalytic Rcat domain, reflecting its release from RING0 (Fig. 3b). The effect of BIO-2007817 was strictly dependent on the presence of pUb; no changes in parkin deuteration were observed upon addition of BIO-2007817 alone (Fig. 3c). The increase in Rcat exposure was also completely blocked by the K211N mutation that prevents pUb binding to RING0 (Fig. 3d) or by substitution of BIO-2007818 with the inactive diastereomer (Fig. 3e). Thus, as observed in the autoubiquitination assays (Fig. 1), the combined action of pUb and BIO-2007817 is required to activate parkin. This occurs through the release of the Rcat domain from its site on RING0 in a manner analogous to activation by parkin phosphorylation29,30,31 (Fig. 3f).

Crystal structure of parkin R0RB:2×pUb complex with BIO-1975900

We turned to crystallography to understand how BIO-2007817 acts as a molecular glue. As crystallization trials with BIO-2007817 were unsuccessful, we expanded the screen to include related compounds with different ethanone substituents. We obtained crystals with the compound BIO-1975900 bound to rat parkin R0RB with two pUb and solved the structure by molecular replacement (Fig. 4; Supplementary Table 2). BIO-1975900 is composed of a central tetrahydropyrazolo-pyrazine core, decorated by a tetrahydroquinoline head and a benzyl and ethanone tail (Fig. 4a). It has roughly 20-fold lower affinity with an EC50 of 2.9 µM in in vitro TR-FRET activity assays compared to 0.15 µM for BIO-200781748….

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