Pitstop‐2 Upsets The Integrity of Nuclear Pore Complexes (NPCs) by Interaction with β‐Propeller Folds of Npc Scaffold Proteins

The small compound Pitstop‐2 is a recent potent inhibitor of clathrin‐mediated endocytosis (CME), widely used in biomedical research areas. In recent years, however, it is observed that it exhibits CME‐independent inhibitory effects on nuclear pore complexes (NPCs), the nucleocytoplasmic gatekeepers. NPCs are elaborate proteinaceous transport nano‐machineries of crucial physiological importance rendering them novel targets for various medical applications. They mediate all nucleocytoplasmic transport forming a physiologically essential selective nucleocytoplasmic barrier. The direct Pitstop‐2 disruptive effects on NPCs manifested themselves at both the structural and functional integrity levels. Moreover, they are massive, acute, and detectable at concentrations equal to CME‐inhibitory concentrations. Pitstop‐2 inhibits CME by binding to the terminal β‐propeller domain of the heavy chain of clathrin. Several NPC scaffold proteins, critical for the structural and functional integrity of the NPC, possess β‐propeller folds. Herein, utilizing computational docking analysis, it is demonstrated that Pitstop‐2 exhibits particularly high binding affinities to β‐propeller folds of NPC scaffold proteins, similar to its binding affinity to the terminal β‐propeller domain of clathrin. The authors, therefore, conclude that Pitstop‐2 is a potent disruptor of NPCs, an activity which, separately or in synergy with CME inhibition, may be exploited for a myriad of pharmacological applications.


Introduction
Clathrin-mediated endocytosis (CME) is an essential gateway of material exchange and communication of a eukaryotic cell with its environment. [1]Its intimate involvement with a multitude of physiologic and pathophysiologic processes has instigated an intensive search for small molecule compounds capable of inhibiting the clathrin-mediated uptake. [2,3]Several candidates have DOI: 10.1002/adbi.202300360been presented in recent years, [4,5] raising hope for diverse potential therapeutic applications in the future, [6][7][8][9] while still lacking the necessary degree of specificity or a defined mechanism of action. [5]Among the most recent additions to the array of inhibitors is Pitstop-2, a compound that has been demonstrated to bind directly to the terminal -propeller domain of the clathrin heavy chain, causing an arrest of coated pit dynamics. [6]14][15][16] The NPC is a modular cylindrical structure with an eight-fold symmetry, comprised of three spoke rings building the NPC scaffold surrounding a central channel, forming the sole gateways for bidirectional nucleocytoplasmic transport (Figure 1a). [18,20]It comprises ∼30 different proteins (nucleoporins or Nups) arranged in multiples of eight which may be classified into three distinct categories. [21]1) Membrane proteins that stably anchor the NPC scaffold to both nuclear membranes.2) Intrinsically disordered and highly flexible Nups with characteristic phenylalanineglycine (FG)-repeat elements that crowd the NPC central channel.They generate the selective permeability barrier function of the NPC. [10,11,22,23]Nup98 is widely considered to be the major barrier-forming FG-Nup. [23,24]3) Architectural Nups, form the NPC scaffold consisting of three spoke rings (cytoplasmic, nucleoplasmic, and an inner ring between those two. [18,20]Scaffold Nups are arranged in Y-shaped complexes termed Y-complexes, coupled together in a head-to-tail manner (Figure 1a). [18,25,26]hey determine the NPC scaffold shape and are essential to link the inner NPC ring to FG-Nups and position them inside the NPC central channel. [27]The Y-complexes contain alpha solenoid and -propeller domains (Figure 1b).The rigid propeller grant the crucial structural stability necessary to keep the highly curved NPC scaffold stably anchored to the nuclear membranes. [21]ased on the substantial similarities in -propeller folds in the heavy chain of clathrin and the Y-complexes and the NPC scaffold, we previously hypothesized that Pitstop-2 might also be able  [6,17] and NPC structure is modified from [13,18] .b) Human NPC scaffold structure based on cryo-electron tomography. [18]The scaffold is made up of Y-shaped subcomplexes, termed Y-complexes.The Nup107/160 subcomplex has 9 members, Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Seh1, and Sec13.The -propellers are highlighted with dotted circles.Nups images are from RCSB PDB (https://www.rcsb.org/structure/7PEQ)and the Y-complex image is modified from [19] .Nups rich in Phenyl-alanine-glycine (FG) domains (FG-Nups) form the stringent NPC barrier inside the channel.Nup98 is the major barrier-maker and interacts with scaffold proteins.
to modulate the barrier and the active nucleocytoplasmic transport functions of the NPCs. [28,29]e reasoned that such similarity might be sufficient for the inhibitor to bind to those Nups and disrupt the structural organization of the NPC scaffold.Our hypothesis was validated by ultrastructural atomic force microscopy (AFM) imaging. [28]Treatment of the nuclear envelopes with the inhibitor has resulted in a disruption of the NPC scaffold to a point where individual Ycomplexes, were no longer arranged along the periphery of the NPC, but were visible also in the proximity of the NPC central channel. [28]These drastic changes in the NPC ultrastructure have caused corresponding alterations in its critical functions. [28]On the one hand, treatment with Pitstop-2 has resulted in a collapse of the nucleocytoplasmic barrier. [28]On the other hand, application of Pitstop-2 prevented binding and translocation of the main transport factor for selective receptor-mediated transport through NPCs, importin-, [22] into the interior of the nucleus. [28]The effects of the inhibitor on the NPC functions were subsequently confirmed in an independent study. [30]
To validate our molecular docking analysis, we started by examining the binding of Pitstop-2 to clathrin, namely the terminal -propeller domain of the heavy chain of clathrin. [6]Consistently with the original publication of Pitstop-2 as a CMEinhibitor, [6] our docking simulation predicted that Pitstop-2 has a high affinity to clathrin (PDB:4G55), with a binding energy of −8.9 Kcal mol −1 , mainly through the formation of hydrogen bridges with ARG64 (3.24 Angstrom, Å) and through hydrophobic interactions with residues ILE52, ILE62, ILE66, LEU82, GLN89 and LEU93 (Figure 2 and Table 1).
We next analyzed the binding interactions between Pitstop-2 and Nup107/160 subcomplex members, which possess propeller folds, Nup37, Nup43, Nup133, Nup120 (yeast analog of human Nup160), Seh1 and Sec13, and used the ß-propellerfree Nup358 from the cytoplasmic NPC filament as a control Nup (Table 1).Our results revealed that Pitstop-2 has a binding energy of −7.5 kcal mol −1 for Nup37.It formed hydrogen bonds with THR295, with a bond length of 2.86 Å, and established hydrophobic contacts with residues GLI199, LEU234, LEU235, ILE291, LEU303, ASN306, and PHE383.Similarly, Pitstop-2 demonstrated a binding energy of −7.0 kcal mol −1 for Nup43, engaging in hydrophobic interactions with residues HIS74, CYS96, GLN114, and THR116, as well as forming two hydrogen bridges with THR94, with bond lengths of 2.8 and 2.98 Å. Pitstop-2 also exhibited a binding energy of −7.9 kcal mol −1 for Nup133, forming hydrogen bridges with ALA446 (bond length: 3.01 Å), ASP449 (bond length: 2.99 Å), and SER468 (bond length: 3.07 Å), and establishing hydrophobic contacts with ILE442, VAL443, and PHE444.In the case of Sec13, Pitstop-2 displayed a binding energy of −7.7 kcal mol −1 , primarily interacting through hydrophobic contacts with residues ILE4, THR26, PHE36, and VAL38.Pitstop-2 exhibited a binding energy of −8.2 kcal mol −1 for Seh1, forming a hydrogen bridge with SER65 at a distance of 2.93 Å and engaging in hydrophobic interactions with ASP62, PRO66, Table 1.Thermodynamic parameters (Gibbs free energy change kcal mol −1 , ΔG°) obtained for the interaction of Pitstop-2 with the terminal -propeller domain of the heavy chain of clathrin, and NPC scaffold Nups members of the Nup107/160 subcomplex with -propellers (Nup37, Nup43, Nup133, Nup120 (yeast Nup, analogous to human Nup160), Seh1 and Sec13).Nup358, part of the cytoplasmic NPC filaments, which has no ß-propellers, acts as a control Nup. and ALA119.The highest binding affinity of Pitstop-2 turned out to be to Nup120, the yeast analogous to the human Nup160, with a binding energy of −8.8 kcal mol −1 , almost equal to the value we obtained for Pitstop-2 target in clathrin.Pitstop-2 engages in hydrophobic interactions with ILE352 and LYS377 in Nup120.Nup358, which is part of the cytoplasmic NPC filaments and has no ß-propellers, acts as a control Nup, [33] and the binding energy we obtained for Pitstop-2 to this Nup358 (−6.4 kcal mol −1 ) was significantly lower than all the other values.
Nup107/160 subcomplex is an essential component of the NPC that is directly involved in maintaining the integrity and stability of the nuclear envelope and facilitating nucleocytoplasmic transport.Our previous study showed that Pitstop-2 could disrupt the NPC permeability barrier, increasing the permeability of the nuclear envelope for 70 kDa dextran and reducing the binding with Imp. [28]We attribute these drastic effects on NPCs' architectural and functional properties to the binding of Pitstop-2 to the -propeller domain of the Nups constituting the Y complexes.
With respect to the observed binding affinity of Pitstop-2 to all six ß-propeller members of the Nup107/160 subcomplex, which is particularly high to Seh1 and Nup160, we cannot yet conclude whether Pitstop-2 binding to the individual scaffold Nups would occur simultaneously or rather in a sequential manner from highest to lowest affinities.However, based on ultrastructural investigations with AFM in our previous work, [28] we can imagine that it binds to all NPC scaffold proteins with ß-propeller folds, considering its massive effect on the NPC scaffold structure, which collapses inwards into the NPC channel entrance upon acute exposure to Pitstop-2.The molecular weight (MW 473.36) of Pitstop-2 may be small enough to enable it to fit into the binding sites of all scaffold Nups with ß-propeller folds, which may account for its rapid and drastic effects on the overall structural integrity of the NPC scaffold. [28]Owing to its highest binding affinities to Nup160 and Seh1, both of which are positioned in the head part of the Nup107/160 subcomplex and are well accessible, we can imagine that Pitstop-2 action starts off at these two binding sites, which are crucial for the head-to-tail arrangement of the Y-complexes and thus structural integrity of the entire scaffold.Binding to Nup160 and Seh1 may rapidly destabilize the entire Nup107/160 subcomplex and expose the other members to further interaction with Pitstop-2, sequentially exacerbating its collective effect on the NPC scaffold.Hence, it is conceivable that the massive effects of Pitstop-2 on the NPC scaffold and overall morphology of the NPC may result from several interaction sites simultaneously or in rapid succession from highest to lowest affinities, and follow-up experimental investigations may provide deeper insights.
Scaffold proteins are necessary to position barrier-forming FG-Nups inside the NPC channel, and FG-Nups are also essential for receptor-mediated transport across NPCs.FG Nups interact with Y-complex components, including Nup96 and Nup107, via hydrophobic binding between phenylalanine residues of FG motifs and Y-complex binding pockets [34] .These interactions contribute to the anchoring and stabilization of FG Nups within the NPC structure.Consequently, disruption of protein-protein interactions in Y-complexes of the NPC scaffold and their interaction partners should have massive effects on NPCs.Notably, our previous data demonstrated that exposure to Pitstop-2 did not induce dissociation of FG-Nups [28] , thereby confirming that Pitstop-2 interferes with the Nup107/160 subcomplex.Consequently, this interference leads to defects in NPC formation and organization, ultimately compromising the integrity of the nuclear envelope.

Conclusion
Pitstop-2 which was originally introduced as a specific inhibitor of clathrin-mediated endocytosis, [6] induces a breakdown of the NPC barrier by a mechanism, independent of CME-inhibition.Moreover, it acts on NPCs at effective concentrations close to those required for significant CME inhibition.
Our proposed mechanism of action for Pitstop-2 disruption of NPCs structural and functional integrity is by direct interference of Pitstop-2 with -propeller folds of Y-subcomplex members.Pitstop-2 can bind with high affinity to -propeller folds of the Y-subcomplex 107/160 members Nup37, Nup43, Nup133, Nup160, Sec13, and Seh1.The binding affinities to Nups are slightly lower or almost equal to its inhibitory binding at the propeller fold of the heavy chain of clathrin, and we, therefore, assume that Pitstop-2 may interfere simultaneously with both CME and NPCs, as concluded in our recent studies. [28,29]Substances capable of reducing the stringency of nucleocytoplasmic permeability barriers could facilitate the development of non-viral systems of intranuclear transgene delivery. [35]By causing a massive rearrangement of the NPC scaffold, Pitstop-2 can compromise the barrier's integrity without dissociating the barrier-forming nucleoporins.Pitstop-2 may be used as a lead substance to design more NPC-specific compounds with no CME-inhibitory activities to enable the intranuclear delivery of therapeutic nanoparticles.Last but not least, NPCs are a linchpin, highly upregulated in transforming and proliferating cancer cells as compared to normal cells, and we propose that Pitstop-2 be considered as a potential anti-cancer drug.

Experimental Section
Molecular Docking: We used the crystallographic structure with the PDB entry code (4G55, 4GQ1, 4I79, 1XKS, 4GQ2, 4GA0, 2PM7, and 3EWE), respectively to Clathrin, Nup37, Nup43, Nup133, Nup120, Nup358, Sec13, and Seh1.For protein preparation, the co-crystallized ligand and water molecules, except for the water molecules that were important in the interaction between the ligand and protein, were eliminated.By Auto Dock Tools 1.5.6 package, [36] all missing hydrogens were added.After calculating Kollman atom charges, nonpolar hydrogens were merged and the file was saved as pdbqt.The ligand, the 3D structures of Pitstop-2 were depicted in Marvin Sketch Ver.5.7, ChemAxon (http://www.chemaxon.com).The active site was predicted using the CASTp3.0server [37] The docking procedure was performed by AutoDock vina [38]

Figure 1 .
Figure 1.Potential effects of Pitstop-2 on NPC scaffold proteins based on their structural similarities to clathrin coat proteins.a) Ultrastructural similarities of the clathrin heavy chain terminal domain (in red) and Nup133 (in green) may allow Pitstop-2 binding to the NPC and disrupting its ultrastructure and functions.Clathrin structures are modified from[6,17] and NPC structure is modified from[13,18] .b) Human NPC scaffold structure based on cryo-electron tomography.[18]The scaffold is made up of Y-shaped subcomplexes, termed Y-complexes.The Nup107/160 subcomplex has 9 members, Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Seh1, and Sec13.The -propellers are highlighted with dotted circles.Nups images are from RCSB PDB (https://www.rcsb.org/structure/7PEQ)and the Y-complex image is modified from[19] .Nups rich in Phenyl-alanine-glycine (FG) domains (FG-Nups) form the stringent NPC barrier inside the channel.Nup98 is the major barrier-maker and interacts with scaffold proteins.