Small molecules targeting endocytic uptake and recycling pathways

Over the past years a growing number of studies highlighted the pivotal role of intracellular trafficking in cell physiology. Among the distinct transport itineraries connecting the endocytic system, both internalization (endocytosis) and recycling (endocytic recycling) pathways were found fundamental to ensure cellular sensing, cell-to-cell communication, cellular division, and collective cell migration in tissue specific-contexts. Consistently, the dysregulation of endocytic trafficking pathways is correlated with several human diseases including both cancers and neurodegeneration. Aimed at suppress specific intracellular trafficking routes involved in disease onset and progression, huge efforts have been made to identify small molecule inhibitors with suitable pharmacological properties for in vivo administration. Here, we review most used drugs and recently discovered small molecules able to block endocytosis and endocytic recycling pathways. We characterize such pharmacological inhibitors by emphasizing their target specificity, molecular affinity, biological activity and efficacy in both in vitro and in vivo experimental models.


Introduction
Endocytic membrane trafficking plays an essential role in delivering both solute molecules and membrane components (e.g., lipids and proteins) to various intracellular destinations (Doherty and McMahon, 2009;Haucke, 2015).Conceptually, endocytic trafficking routes are relatively simple, with the main pathways carrying either to degradation in lysosomes or to recycle back to the plasma membrane.However, genetic redundancy and pleiotropy profoundly impact on molecular organization of membrane trafficking, thus limiting identification of both specialized trafficking itineraries and pivotal protein interactions (Yarwood et al., 2020).
Endocytic membrane trafficking is a ubiquitous process in eucaryotic organisms.Nonetheless, not all cells respond to perturbation of membrane trafficking machinery in the same way (De Matteis and Luini, 2011;Yarwood et al., 2020;García-Cazorla et al., 2022).This is posited to depend on: 1) the abundance and the degree of functional redundancy of membrane trafficking components, and 2) the relevance of the transported cargo for cellular functions and tissue homeostasis.For instance, the nervous system is susceptible to disruption of endocytic genes involved in both endocytic recycling and autophagy.This is due to both the low proliferating activity and the elevated speed of neurotransmitters secretion/internalization that characterize neuronal cells (Schreij et al., 2016).Consequently, impairment in endocytic trafficking pathways significantly affect behaviour and function of the nervous tissue, often resulting in neurological disorders including frontotemporal dementia (FTD), Alzheimer and Parkinson diseases (Harold et al., 2009;DeJesus-Hernandez et al., 2011;Vilariño-Güell et al., 2011;Vilariño-Güell et al., 2014).Similarly, alteration in endocytic recycling significantly impact in cells with elevated secretory activity, such as pancreatic beta cells.In particular, mutations in AS160, an endocytic recycling regulator, impairs the translocation of the glucose transporter GLUT4, thus leading to defective glucose blood clearance and hence muscle insulin-resistance and type 2 diabetes (Karlsson et al., 2005;Mîinea et al., 2005;Moltke et al., 2014).Despite these considerations, the employment of membrane trafficking alterations to predict both type and status of a disease is far from being achieved.
The therapeutic targeting of membrane trafficking pathways might be used to increase both delivery and efficacy of currently employed therapeutics for cancer, respiratory disorders and, concomitantly, to provide novel strategies for the treatment of over 300 Mendelian genetic diseases (Chew et al., 2020;Kang et al., 2020;Riva et al., 2020;García-Cazorla et al., 2022).Unfortunately, the complex organization of the endomembrane system limits the identification of membrane trafficking inhibitors using phenotype-based screening strategies.In parallel, it remains still difficult to identify druggable molecular mechanisms pivotal for transport of specific molecular cargoes (De Matteis et al., 2013;Mishev et al., 2013).
This review provides an overview of main drugs controlling regulatory functions of endocytic proteins.This is particularly important in view of recent progress into endocytic trafficking field.To get insights into mechanisms characterizing novel transport itineraries that are not covered by this review, we remand readers to these excellent works (Redpath et al., 2020;Renard and Boucrot, 2021;Sigismund et al., 2021;Gilleron and Zeigerer, 2023).

Targets for endocytic recycling
The internalization and their transport back to the plasma membrane of both solute molecules and membrane components (e.g., lipids and proteins) is carryout by both the endocytic and the recycling membrane trafficking machineries, respectively.Every transport itinerary requires the budding, scission and transport of vesicles from the donor compartment and their subsequent targeting, tethering, docking and fusion to the acceptor membrane.Notably, these trafficking steps are controlled by protein subsets that, according to their roles during the transport process, can be organized into specific functional modules.Below, we provide description of building blocks used by both endocytic and recycling pathways.This list encompass both adaptors, coats, shaping, fission, small GTPase, kinase, tethering and fusion proteins involved in endocytic recycling (Liberali et al., 2008).

Shaping module
Bin/amphiphysin/Rvs (BAR) domain-containing proteins play a prominent role in membrane remodelling in response to protein surface density, membrane tension, or membrane shape alterations.Arfaptin, Amphiphysin, Sortin Nexins, Endophilin, FCHo proteins are the most pervasive membrane-shaping regulators controlling the invagination of both cell surface and endocytic membranes.In addition to the BAR domain, such membrane remodelling proteins can encode a small GTPase-regulatory region.For instance, ASAP and ACAP proteins display an Arf-GAP domain which allow functional coupling between BAR-mediated membrane sculpting and actin polymerization, specifically.Lastly, while the mentioned proteins control endocytic vesicle formation on both endosome and plasma membrane, a different BAR domaincontaining proteins family (e.g., MIM, IRTKS, IRSp53) evolved to direct the generation of plasma membrane protrusion (Simunovic et al., 2015;Renard et al., 2018;Simunovic et al., 2019).

Fission module
The membrane fission module relies on mechanoenzymatic machineries that bind phosphatidylinositol-enriched membranes to favour membrane constriction through a GTP-dependent mechanism.Dynamin proteins (e.g., DNM1, DNM2, DNM3) are GTPase enzymes that hydrolyse GTP to prompt vesicle fissions in endocytic membrane-bound compartments.DNM1 and DNM3 are enriched in brain and they both mediate retrieval of synaptic vesicle membranes, an event that occurs in concomitance with exocytic fusion and cargo recycling to the cell surface.In contrast, DNM2 is ubiquitously expressed and mediates endocytic uptake (Ferguson and De Camilli, 2012;Wu et al., 2014;Lee et al., 2016;Renard et al., 2018;Jimah and Hinshaw, 2019;Imoto et al., 2022).

Rab GTPase modules
The RabGTPase module comprises over 60 distinct genes in the human genome.Rab GTPases are molecular switches that, by cycling between an active and inactive state, they recruit, on membranes, specific molecular effectors.Through their effectors, Rab GTPases regulate vesicle formation, vesicle movement along actin and tubulin networks, and membrane fusion.Notably, specific Rab GTPases subsets are localized to different membrane-bound compartments and hence they act as signpost to recognize transport itineraries in mammalian cells.In this context, RAB4 family (A, B, C), RAB5 family (A, B, C), RAB13, RAB 20, RAB21, RAB22 family (A, B), and RAB23 are restricted to early endosomes, while the recycling compartment stained positive for RAB3 family (A, B, C, D), RAB8 family (A, B), RAB10, RAB11 family (A, B, C/RAB25), RAB14, RAB15, RAB17, RAB35 (Wandinger-Ness and Zerial, 2014).

Fusion module
The fusion module comprises SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)motif containing proteins that mediate vesicles fusion to acceptor membranes.Previously named as v-and t-SNARE, due to their localization to either vesicle or target membranes, while currently classified into Qa-, Qb, Qc-and R-SNARE based on their sequence features, SNARE proteins bind to each other to form a parallel fourhelix bundle.This helix structure, bridges and ultimately fuses vesicle membranes.The endocytic fusion machinery requires Syntaxin (Stx), VAMP and SNAP-25 protein homologues such as: Stx13, Stx6, Stx2, Stx4, Vti1a, VAMP4, and VAMP7.Targeting specificity is determined by the combinatorial assembly of the over 35 different SNAREs.Nonetheless, recent reports indicate that some SNAREs can functionally substitute for each other if they belong to the same subfamily (Jahn and Scheller, 2006;Koike and Jahn, 2017;Koike and Jahn, 2019).
In the section below, we describe main endocytic inhibitors characterized by elevated target specificity.In this summary, we not

Clathrin heavy chain inhibitors
Clathrin heavy chain (CHC) is a self-assembling protein that coats transport vesicles during their endocytic sorting.The assembly of clathrin coat requires interaction between CHC and clathrin-associated sorting proteins (CLASPs) through the CHC terminal domain (TD) (Schmid and McMahon, 2007;Ranjan et al., 2017).CLASPs comprise many endocytic proteins such as the AP2 complex, AP180, CALM, Eps15, Amphiphysin involved in internalization of many growth factor receptors (Lemmon and Traub, 2012).CLASPs localization guides clathrin basket formation at distinct cellular compartments, allowing directionality in membrane trafficking flow.As an example, clathrin-coated vesicles (CCVs), programmed for being delivered from cell surface and internal organelles, are assembled on both endosomes and plasma membrane by specific adaptor proteins such as AP1B1 (designated β1) and AP2B2 (β2), respectively.Based on the important role played by CHC and CLASPs binding for CCVs assembly, huge efforts were made to identify compounds able to block the interaction between CHC's TD and CLASPs, of which Pitstop2 and Endosidin9-17 are the most noticeable.
Pitstop2 associates with the clathrin TD and obstructs binding of accessory proteins involved in both maturation and disassembly of CCP such as Amphysin, AP180, Synaptojanin, OCRL (von Kleist et al., 2011).In cells, Pitstop2 blocks clathrin-mediated endocytosis and generates enlarged endosomes when used in rodents and specifically at the calyx of Held, a synapse optimized for high frequency synaptic transmission in the auditory brainstem (Paksoy et al., 2022).Nonetheless, to date several clathrinindependent effects were reported for Pitstop2 (Dutta et al., 2012).
A similar mechanism of function is employed by Endosidin9 (ES9), a small molecule that induces, after short exposure (~30 min), both clathrin and AP2 mislocalization in human, plants and fruit-fly cells (Dejonghe et al., 2016).Notably, ES9-17, a ES9 more potent derivative, was demonstrated to block endocytosis in Arabidopsis Thaliana, a model system resistant to Pitstop2 inhibition due to an aminoacidic substitution (residue 80) in plant's clathrin heavy chain isoform.

Caveolin inhibitors
Caveolins are integral membrane proteins that play an important structural role in caveolae-mediated endocytosis, an endocytic process mediating the internalization of Cholera toxin, Ebola, Hepatitis B, Japanese encephalitis, human coronaviruses -229E and -OC43 (Xing et al., 2020;Parton et al., 2021).Three distinct caveolin isoforms were identified (Cav1, Cav2, Cav3) and found differentially expressed in human tissues.In particular, Cav1 is enriched in brain, skeletal muscle, liver, stomach, lung, kidney and heart.Cav2 is predominantly expressed in endothelial cells, smooth muscle cells, and fibroblasts, while Cav three is found in muscular tissue (Scherer et al., 1997;McMahon et al., 2009).Caveolins are composed by a transmembrane region flanked by two (N-, C-) terminal domains, each of them is exposed to cell's cytoplasmic side and involved in caveolin posttransaltional regulation.The transmembrane portion comprises an oligomerization domain and a scaffolding domain which control, respectively, caveolin oligomerization and cholesterol binding (Parton and del Pozo, 2013;Porta et al., 2022).Both these proteinprotein and protein-lipid interactions are required for caveolinmediated caveolae formation.The oligomerization domain mediates the aggregation of caveolin in 14-16-unit oligomers (Porta et al., 2022), while the scaffolding portion allows both membrane insertion into cholesterol-enriched membrane regions and interaction with cholesterol-binding proteins (Krishna and Sengupta, 2019).
Currently, two distinct strategies are available to block caveolin activity during endocytosis: cholesterol inhibition and caveolin-oligomer disruption.The first employs Methyl-bcyclodextrin, FilipinIII and Nyastatin to deplete cholesterol and ergosterol from cellular membranes, while the second uses WL47, a synthetic peptide, to reduce in-vitro caveolin oligomers assembly (Plummer and Manchester, 2013;Gilliam et al., 2016).

CLASPs inhibitors
The interaction between clathrin-associated sorting proteins (CLASPs) and clathrin guides the internalization of cell surface proteins.Both β-arrestins and AP2 complex bind clathrin and both are required to induce internalization of cell surface receptors.Specifically, β-arrestins are activated downstream G-protein coupled receptors (GPCRs).Following sustained agonist stimulation, β-arrestin are recruited at the plasma membrane by phosphorylated-activated GPCRs.This event promotes functionally uncoupling of the activated receptors from their heterotrimeric G proteins (Lohse et al., 1990).Subsequently, the association of βarrestins to AP2 induces GPCR internalization and trafficking towards endosomes for their degradation/recycling.
Barbadin is a small molecule that targets the contact interface between β-arrestin and AP2.Barbadin blocks GPCR internalization without affecting trafficking of Transferrin receptor (TfR), a non-GPCR that interacts directly with AP2 in a β-arrestin independent manner (Jing et al., 1990).In a preclinical experiment, Barbadin was demonstrated to potentiate the effects of Lorcaserin, a serotonin 2C receptor (5-HT2CR) selective agonist.Barbadin treatment inhibits 5-HT2CR internalization after lorcaserin stimulation, maintaining proper proopiomelanocortin (POMC) neuron responses to serotonin-agonist challenge in vivo, ultimately leading to appetite reduction and weight gain in mouse models (He et al., 2021).

Dynamin inhibitors
Dynamins are large GTPase proteins that mediate membrane fission and fusion during endocytosis, recycling, and organelle biogenesis (Jimah and Hinshaw, 2019).Three distinct dynamin isoforms were identified and found differentially expressed in human tissues.Dynamin I is mostly expressed in brain-related tissues, Dynamin II is ubiquitous, whereas Dynamin III is enriched in brain, lungs, and testis (Ferguson and De Camilli, 2012).All dynamin isoforms contain a GTPase (G) domain involved in GTP binding and hydrolyses, a pleckstrin-homology (PH) domain which recognizes PtdIns(4,5)P2, a membrane lipid, and a GTPase effector domain (GED) that allows oligomerization and stimulation of Dynamin GTPase activity (Jimah and Hinshaw, 2019).Three distinct classes of small molecule inhibitors were developed to differentially target either dynamin's oligomerization, GTPase activity or lipid-stimulated GTPase activity (Table 1).
Dyngo-4A and Dynasore are small molecules that, by binding allosteric sites in the Dynamin G domain, block dynamin oligomerization into either ring-like or helical structures, thereby impeding cooperative GTP hydrolysis, membrane tubulation and hence clathrin-mediated endocytosis, as demonstrated by reduction of TfR uptake (Tuma and Collins, 1994;Hinshaw and Schmid, 1995;Macia et al., 2006;McCluskey et al., 2013).A different mechanism is adopted by Chlorpromazine, a phenothiazine-derived antipsychotic drug and MiTMAB.These compounds block lipid-stimulated Dynamin GTPase activity by competing with lipid binding (Daniel et al., 2015).In parallel, competitive inhibition of GTP nucleotide binding is used by Pthaladyn-23 (Odell et al., 2010).
One of the potential employments of dynamin inhibitors is the boost of antibody-dependent cellular cytotoxicity (ADCC) response for cancer treatment.By blocking internalization of cell surface antigens, dynamin inhibitors increase target receptor availability for antibody binding.Thus, retention of antigen-antibody complexes enhances recognition of cancer cells by natural killer (NK) cells.These findings were corroborated in preclinical experiments by using Dyngo-4a and prochloropyrazine, a less toxic chlorpromazine analogue.These dynamin inhibitors were found effective in combination with cetuximab, trastuzumab and avelumab, three distinct clinical-approved antibody-based treatment targeting EGFR, HER2 and PD-L1 receptors, respectively (Chew et al., 2020).Although impressive results were obtained by these compounds, dynamin inhibitors are not isoform-selective and hence not able to block specific trafficking pathways.Since dynamin isoforms act on distinct cellular districts (Gray et al., 2003), major concerns regarding their ontarget toxicity during endocytic recycling are present.

Kinase inhibitors
Kinases are enzymes that catalyse the transfer of phosphate groups to either proteins or lipids.As a result, kinases activity is rarely limited to endocytic pathways, often resulting in perturbation of several cellular processes (Roskoski, 2015).Nonetheless, the kinase-induced phosphorylation mechanism permits the endocytic machinery to react to changing cellular demands and hence ensuring appropriate cell's response to a fluctuating environment (Roskoski, 2015).

Small molecules targeting the endocytic recycling machinery
Delivery of endocytosed plasma membrane components to various intracellular compartments is achieved by a multitude of trafficking pathways.Based on the final cargo's destination, such intracellular trafficking routes are classified in the degradative, retrograde and recycling routes (Scott et al., 2014).In this section, we describe lysosomotropic agents (e.g., bafilomycin A1, NH4Cl, chloroquine, ionomycin, nigericin, monensin) and small molecules affecting endocytic recycling (Johnson et al., 1993;Scott and Gruenberg, 2011) (Supplementary Figure S2; Table 2).

Lysosomotropic inhibitors
Many compounds belonging to the family of antimalarial agents, such as chloroquine and primaquine, were introduced as recycling inhibitors (van Weert et al., 1995;van Weert et al., 2000).These small molecules are weak bases, that once protonated, accumulate in endosomes causing both neutralization and deacidification of endosomal pH, thus resulting in endosomal recycling inhibition.
Primaquine and chloroquine are antiviral and antiparasite compounds used to block Zika, Ebola and malaria-related infections (Naghipour et al., 2020;Persoons et al., 2021).Moreover, primaquine and Chloroquine were recently under evaluation for therapeutic treatment of autoimmune diseases (Rainsford et al., 2015).As generic medications, huge efforts have been made to repurpose Chloroquine and Primaquine (and all others lysosomotropic drug) for cancer treatment.However, results coming from these studies are puzzling, thus limiting the employment of lysosomotropic drugs in both basic and clinical research (Ashley et al., 2014).Similar consideration must be made for BafilomycinA1, a macrolide antibiotic that blocks receptor recycling by inhibiting V-ATPase proton pump (Yoshimori et al., 1991).The V-ATPase consists of two main multisubunit complexes named V0 and V1.Structural studies and biochemical experiments defined that BafilomycinA1 causes steric hindrance between elements of V0 subunit, resulting in inhibition of V-ATPase activity (Wang et al., 2021).
In contrast to the protein kinases, members of the lipid kinase family have less structural similarity in enzyme's active site, simplifying generation of isoform-selective inhibitors (Roskoski, 2016).As an example, reduction of 5′ phosphorylated phosphoinositide signalling can be obtained using both competitive and non-competitive ATP inhibitors.Apilimod, an ATP competitive inhibitor, and NIH-12848, a non-competitive ATP inhibitor, reduce endocytic recycling of internalized cargoes by blocking the activity of PIKfyve and PI5P4Kγ lipid kinases, respectively (Clarke et al., 2015;Sbrissa et al., 2018;Baranov et al., 2020).Despite these findings, most studies are currently focused on the role of both Apilimod and NIH-12848 in endolysosomal regulation and its impact in immune-regulatory associated diseases (Gayle et al., 2017;Poli et al., 2021).Whether the endocytic recycling and the lysosomal trafficking pathways are linked by the 5′ phosphorylated phosphoinositide signalling is still unclear.

Small GTPase inhibitors
Small GTPases are enzymes that catalyse the hydrolysis of guanosine triphosphate (GTP) and the subsequent generation of guanosine diphosphate (GDP).Activation of small GTPase is controlled by guanine nucleotide exchange factors (GEFs) which promote the release of GDP and the subseqeunt GTP loading.Conversely, GTPase-activating proteins (GAPs) bind to activated small GTPase to stimulate their GTPase activity.Small GTPase targeting might be used to control main steps involved in membrane trafficking including vesicle generation, transport, and fusion.Historically, the evidence of small GTPase targeting potential in membrane transport was demonstrated by Brefeldin A (BFA), a fungal toxin able to block ARF small GTPase activity (Helms and Rothman, 1992;Reiner and Lundquist, 2018).BFA is a protein-protein interaction inhibitor that disrupts the interaction between Arf1 and GBF1, an Arf GEF (Rouhana et al., 2013).Optimization of this proteinprotein inhibitor has led to the development of LG186.LG186 is a selective Arf1-GBF1 interaction inhibitor, used to block, in addition to common Golgi-localized ARF1 trafficking pathways, CLIC/GEEC internalization (Klausner et al., 1992;Boal et al., 2010;Sathe et al., 2018).
However, the targeting of small GTPase by small molecule inhibitors remains difficult (Gray et al., 2020).At this regard, novel approaches, that use synthetic peptides and covalent small molecules, are now employed to prevent the interaction between small GTPase and their effector proteins and hence small GTPase signalling (Ali et al., 2019).As an example, the stapled peptide RFP14 blocks the interaction between Rab25 and FIP2, a Rab25 effector protein, causing a decrease of RAB25-driven cell proliferation (Mitra et al., 2017).A similar approach was employed to achieve RAB27 pathway inhibition, a key player in exosome secretion.Nexinhib20, a small molecule targeting the interaction between Rab27a and its effector JFC1, was found to regulate exocytosis-dependent neutrophil's function and exosome secretion both in vitro and in vivo models of inflammation, a context in which exosome secretion and recycling are pivotal pathways for cancer disease progression (Johnson et al., 2016).Lastly, by taking advantage of two residues, that are unique to Rab27A and Rab27B, among the over 60 Rab family proteins (i.e., C123 and C188), a recent report identifies two covalent ligands (A01 and B01) that react preferentially with these cysteines, paving the way for future development of covalent RAB27A signalling inhibitors (Jamshidiha et al., 2022).

Conclusion
The pharmacological targeting of both endocytosis and endosomal recycling pathways has the potential to improve therapeutic treatment of Mendelian genetic diseases, parasites/ virus infections, neurodegeneration and cancers.However, due to the pleiotropic behaviour of endocytic genes, it remains difficult to hijack specific transport itineraries, as observed for Endosidin 2, a Exo70 small molecule inhibitor, that affects both constitutive exocytosis and receptor recycling.In parallel, drug repurposing has been explored to evaluate the therapeutic potential of membrane trafficking targeting in human diseases.For instance, Chlorpromazine, a GPCR antagonist used as an antipsychotic medication, was found to block dynamins activity and hence endocytosis of surface proteins.Despite these considerations, the druggability of many trafficking molecules is still unexplored.
In recent years, the development of both virtual screenings and cell-based functional assays have been employed for identification of novel trafficking inhibitors with improved target specificity and pathways selectivity.In this context, synthetic peptides, or covalent inhibitors able to block Rab small GTPases function could address selectivity issues in membrane trafficking inhibition.

TABLE 1
Small molecules inhibitors targeting endocytic molecules.

TABLE 2
Small molecules inhibitors targeting specific recycling modules.