Pharmacological Chaperones Research Articles

Multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease

A concise asymmetric synthesis of clickable enantiomeric pyrrolidines was achieved using Crabbé-Ma allenation. The synthesized iminosugars were grafted by copper–free strain-promoted alkyne-azide cycloaddition onto phosphorus dendrimers. The hexavalent and dodecavalent pyrrolidines were evaluated as β–glucocerebrosidase inhibitors. The level of inhibition suggests that monofluorocyclooctatriazole group may contribute to the affinity for the protein leading to potent multivalent inhibitors. Docking studies were carried out to rationalize these results. Then, the iminosugars clusters were evaluated as pharmacological chaperones in Gaucher patients’ fibroblasts. An increase in β–glucocerebrosidase activity was observed with hexavalent and dodecavalent pyrrolidines at concentrations as low as 1 µM and 0.1 µM, respectively. These iminosugar clusters constitute the first example of multivalent pyrrolidines acting as pharmacological chaperones against Gaucher disease.

Exploring ligand interactions with human phosphomannomutases using recombinant bacterial thermal shift assay and biochemical validation

PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of phosphomannomutase-1 to enhance intracellular glucose-1,6-bisphosphate have been proposed as therapeutical approaches.We used Recombinant Bacterial Thermal Shift Assay to assess the binding of a substrate analog to phosphomannomutase-2 and the specific binding to phosphomannomutase-1 of an FDA-approved drug - clodronate. We also deepened the clodronate binding by enzyme activity assays and in silico docking. Our results confirmed the selective binding of clodronate to phosphomannomutase-1 and shed light on such binding.

N‐Methyl‐N‐Alkylaminocyclopentanes: Powerful and Selective β‐d‐Glucocerebrosidase Inhibitors

AbstractBuilding upon a previously established (2+3)‐cycloaddition strategy, a series of N,N‐dialkylated aminocyclopentanes was synthesized using a partially protected eno‐furanose as the starting point. The resulting N‐methylisoxazolidine was subsequently transformed into the corresponding aminocyclopentane, which was further N‐alkylated, yielding a collection of compounds with potential as inhibitors and pharmacological chaperones of β‐d‐glucocerebrosidase. A comprehensive screening involving a range of biologically relevant glycosidases unveiled that these compounds exhibit remarkable potency and selectivity as inhibitors of human lysosomal β‐d‐glucocerebrosidase. However, none of these compounds exhibit significant activity enhancement of Morbus Gaucher related p.N409S/p.L483P mutant β‐d‐glucocerebrosidase.

Mechanistic Insights into Dibasic Iminosugars as pH-Selective Pharmacological Chaperones to Stabilize Human α-Galactosidase.

The use of pharmacological chaperones (PCs) to stabilize specific enzymes and impart a therapeutic benefit is an emerging strategy in drug discovery. However, designing molecules that can bind optimally to their targets at physiological pH remains a major challenge. Our previous study found that dibasic polyhydroxylated pyrrolidine 5 exhibited superior pH-selective inhibitory activity and chaperoning activity for human α-galactosidase A (α-Gal A) compared with its monobasic parent molecule, 4. To further investigate the role of different C-2 moieties on the pH-selectivity and protecting effects of these compounds, we designed and synthesized a library of monobasic and dibasic iminosugars, screened them for α-Gal A-stabilizing activity using thermal shift and heat-induced denaturation assays, and characterized the mechanistic basis for this stabilization using X-ray crystallography and binding assays. We noted that the dibasic iminosugars 5 and 20 protect α-Gal A from denaturation and inactivation at lower concentrations than monobasic or other N-substituted derivatives; a finding attributed to the nitrogen on the C-2 methylene of 5 and 20, which forms the bifurcated salt bridges (BSBs) with two carboxyl residues, E203 and D231. Additionally, the formation of BSBs at pH 7.0 and the electrostatic repulsion between the vicinal ammonium cations of dibasic iminosugars at pH 4.5 are responsible for their pH-selective binding to α-Gal A. Moreover, compounds 5 and 20 demonstrated promising results in improving enzyme replacement therapy and exhibited significant chaperoning effects in Fabry cells. These findings suggest amino-iminosugars 5 and 20 as useful models to demonstrate how an additional exocyclic amino group can improve their pH-selectivity and protecting effects, providing new insights for the design of pH-selective PCs.

Exploring the efficacy and safety of Ambroxol in Gaucher disease: an overview of clinical studies.

Gaucher disease (GD) is mainly caused by glucocerebrosidase (GCase) enzyme deficiency due to genetic variations in the GBA1 gene leading to the toxic accumulation of sphingolipids in various organs, which causes symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, and neurological manifestations. GD is clinically classified into the non-neuronopathic type 1, and the acute and chronic neuronopathic forms, types 2 and 3, respectively. In addition to the current approved GD medications, the repurposing of Ambroxol (ABX) has emerged as a prospective enzyme enhancement therapy option showing its potential to enhance mutated GCase activity and reduce glucosylceramide accumulation in GD-affected tissues of different GBA1 genotypes. The variability in response to ABX varies across different variants, highlighting the diversity in patients' therapeutic outcomes. Its oral availability and safety profile make it an attractive option, particularly for patients with neurological manifestations. Clinical trials are essential to explore further ABX's potential as a therapeutic medication for GD to encourage pharmaceutical companies' investment in its development. This review highlights the potential of ABX as a pharmacological chaperone therapy for GD and stresses the importance of addressing response variability in clinical studies to improve the management of this rare and complex disorder.

Switch from enzyme replacement therapy to pharmacologic chaperone: Improvement in advanced Fabry nephropathy

Switch from enzyme replacement therapy to pharmacologic chaperone: Improvement in advanced Fabry nephropathy

Initiation of pharmacologic chaperone therapy for Fabry disease in the Canadian Fabry Disease Initiative (CFDI) registry is not associated with reduction of kidney function

Initiation of pharmacologic chaperone therapy for Fabry disease in the Canadian Fabry Disease Initiative (CFDI) registry is not associated with reduction of kidney function

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction.

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the β-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on β-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

Carriers of Heterozygous Loss-of-Function ACE Mutations Are at Risk for Alzheimer's Disease.

We hypothesized that subjects with heterozygous loss-of-function (LoF) ACE mutations are at risk for Alzheimer's disease because amyloid Aβ42, a primary component of the protein aggregates that accumulate in the brains of AD patients, is cleaved by ACE (angiotensin I-converting enzyme). Thus, decreased ACE activity in the brain, either due to genetic mutation or the effects of ACE inhibitors, could be a risk factor for AD. To explore this hypothesis in the current study, existing SNP databases were analyzed for LoF ACE mutations using four predicting tools, including PolyPhen-2, and compared with the topology of known ACE mutations already associated with AD. The combined frequency of >400 of these LoF-damaging ACE mutations in the general population is quite significant-up to 5%-comparable to the frequency of AD in the population > 70 y.o., which indicates that the contribution of low ACE in the development of AD could be under appreciated. Our analysis suggests several mechanisms by which ACE mutations may be associated with Alzheimer's disease. Systematic analysis of blood ACE levels in patients with all ACE mutations is likely to have clinical significance because available sequencing data will help detect persons with increased risk of late-onset Alzheimer's disease. Patients with transport-deficient ACE mutations (about 20% of damaging ACE mutations) may benefit from preventive or therapeutic treatment with a combination of chemical and pharmacological (e.g., centrally acting ACE inhibitors) chaperones and proteosome inhibitors to restore impaired surface ACE expression, as was shown previously by our group for another transport-deficient ACE mutation-Q1069R.

Genetic and Pharmacological Modulation of Cellular Proteostasis Leads to Partial Functional Rescue of Homocystinuria-Causing Cystathionine-Beta Synthase Variants.

Homocystinuria (HCU), an inherited metabolic disorder caused by lack of cystathionine beta-synthase (CBS) activity, is chiefly caused by misfolding of single amino acid residue missense pathogenic variants. Previous studies showed that chemical, pharmacological chaperones or proteasome inhibitors could rescue function of multiple pathogenic CBS variants; however, the underlying mechanisms remain poorly understood. Using Chinese hamster DON fibroblasts devoid of CBS and stably overexpressing human WT or mutant CBS, we showed that expression of pathogenic CBS variant mostly dysregulates gene expression of small heat shock proteins HSPB3 and HSPB8 and members of HSP40 family. Endoplasmic reticulum stress sensor BiP was found upregulated with CBS I278T variant associated with proteasomes suggesting proteotoxic stress and degradation of misfolded CBS. Co-expression of the main effector HSP70 or master regulator HSF1 rescued steady-state levels of CBS I278T and R125Q variants with partial functional rescue of the latter. Pharmacological proteostasis modulators partially rescued expression and activity of CBS R125Q likely due to reduced proteotoxic stress as indicated by decreased BiP levels and promotion of refolding as indicated by induction of HSP70. In conclusion, targeted manipulation of cellular proteostasis may represent a viable therapeutic approach for the permissive pathogenic CBS variants causing HCU.

Elucidating the role of the AD risk gene SORL1 in hiPSC‐derived microglial cells

AbstractBackgroundhiPSC models, from which all cell types of the CNS can be generated, have provided key insights into cell‐type specific processes implicated by AD genetics. Endo‐lysosomal network (ELN) trafficking is a biological pathway implicated in AD and the endosomal receptor SORL1 is strongly associated with AD risk, however studies of SORL1 function in glial cells are lacking. Because ELN trafficking is highly related to neuroimmune function, we modeled SORL1 deficiency in hiPSC‐derived microglia and tested whether pharmacologic or genetic rescue with components of retromer improve cellular phenotypes.MethodWe used an established isogenic series of hiPSC lines that harbor deficiencies in SORL1 (SORL1 KO or SORL1 +/‐) or missense variants in SORL1 (SORL1Var) associated with AD. We differentiated microglial‐like cells (hiPSC‐MGLs) and used cellular assays that examined the function of the ELN. In hiPSC‐MGLs we measured phagocytosis, lysosome function, and pro‐inflammatory cytokine release. For rescue experiments, we treated cells with TPT‐260, a pharmacologic chaperone that stabilizes retromer, and lentiviral vectors expressing retromer subunits.ResultIn SORL1 deficient and SORL1 variant hiPSC‐MGLs, we document ELN phenotypes that converge on the lysosome, including lysosomal swelling and impaired degradation. Interestingly, we also document decreased lysosomal exocytosis, a calcium‐regulated process by which immune cells release lysosomal enzymes and undegraded substrates to the extracellular space to aid in cellular homeostasis, immune‐related signaling and tissue remodeling. In SORL1 deficient cells, lysosomal impairments were observed with abnormal neuroimmune function including differential expression and secretion of cytokines, in response to pro‐inflammatory stimuli Our preliminary data suggests that enhancement of retromer function, either genetically or pharmacologically, can improve these phenotypes.ConclusionUsing an hiPSC‐based model, we show that we can uncover dynamic phenotypes that are linked to AD risk genes. Mutations in SORL1 are now frequently discovered in early‐onset AD patients and the SORL1 locus has a strong genome‐wide signal for late‐onset AD. Together, this suggests that therapeutic strategies targeting the SORL1‐retromer pathway are greatly needed. In this study, we elucidate a role for the SORL1‐retromer pathway in neuroimmune function and provide validation that enhancing this pathway can improve cell‐type specific AD phenotypes in a human model.

Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small-molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase and demonstrated that CFTR-F508del ERAD is robust. Gene-drug interaction experiments illustrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.

Validation of a highly sensitive HaloTag-based assay to evaluate the potency of a novel class of allosteric β-Galactosidase correctors.

Site-directed Enzyme Enhancement Therapy (SEE-Tx®) technology is a disease-agnostic drug discovery tool that can be applied to any protein target of interest with a known three-dimensional structure. We used this proprietary technology to identify and characterize the therapeutic potential of structurally targeted allosteric regulators (STARs) of the lysosomal hydrolase β-galactosidase (β-Gal), which is deficient due to gene mutations in galactosidase beta 1 (GLB1)-related lysosomal storage disorders (LSDs). The biochemical HaloTag cleavage assay was used to monitor the delivery of wildtype (WT) β-Gal and four disease-related β-Gal variants (p.Ile51Thr, p.Arg59His, p.Arg201Cys and p.Trp273Leu) in the presence and absence of two identified STAR compounds. In addition, the ability of STARs to reduce toxic substrate was assessed in a canine fibroblast cell model. In contrast to the competitive pharmacological chaperone N-nonyl-deoxygalactonojirimycin (NN-DGJ), the two identified STAR compounds stabilized and substantially enhanced the lysosomal transport of wildtype enzyme and disease-causing β-Gal variants. In addition, the two STAR compounds reduced the intracellular accumulation of exogenous GM1 ganglioside, an effect not observed with the competitive chaperone NN-DGJ. This proof-of-concept study demonstrates that the SEE-Tx® platform is a rapid and cost-effective drug discovery tool for identifying STARs for the treatment of LSDs. In addition, the HaloTag assay developed in our lab has proved valuable in investigating the effect of STARs in promoting enzyme transport and lysosomal delivery. Automatization and upscaling of this assay would be beneficial for screening STARs as part of the drug discovery process.

PH-Responsive Trihydroxylated Piperidines Rescue The Glucocerebrosidase Activity in Human Fibroblasts Bearing The Neuronopathic Gaucher-Related L444P/L444P Mutations in GBA1 Gene.

Engineering bioactive iminosugars with pH-responsive groups is an emerging approach to develop pharmacological chaperones (PCs) able to improve lysosomal trafficking and enzymatic activity rescue of mutated enzymes. The use of inexpensive l-malic acid allowed introduction of orthoester units into the lipophilic chain of an enantiomerically pure iminosugar affording only two diastereoisomers contrary to previous related studies. The iminosugar was prepared stereoselectively from the chiral pool (d-mannose) and chosen as the lead bioactive compound, to develop novel candidates for restoring the lysosomal enzyme glucocerebrosidase (GCase) activity. The stability of orthoester-appended iminosugars was studied by 1 H NMR spectroscopy both in neutral and acidic environments, and the loss of inhibitory activity with time in acid medium was demonstrated on cell lysates. Moreover, the ability to rescue GCase activity in the lysosomes as the result of a chaperoning effect was explored. A remarkable pharmacological chaperone activity was measured in fibroblasts hosting the homozygous L444P/L444P mutation, a cell line resistant to most PCs, besides the more commonly responding N370S mutation.

Identification of novel glucocerebrosidase chaperones by unexpected skeletal rearrangement reaction

Compound 5 was identified from a high-throughput screening campaign as a small molecule pharmacological chaperone of glucocerebrocidase (GCase), a lysosomal hydrolase encoded by the GBA1 gene, variants of which are associated with Gaucher disease and Parkinson’s disease. Further investigations revealed that compound 5 was slowly transformed into a regio-isomeric compound (6) in PBS buffer, plausibly via a ring-opening at hemiaminal moiety accompanied by subsequent intramolecular CC bond formation. Utilising this unexpected skeletal rearrangement reaction, a series of compound 6 analogues was synthesized which yielded multiple potent GCase pharmacological chaperones with sub-micromolar EC50 values as exemplified by compound 38 (EC50 = 0.14 μM).

OR03-01 Functional Rescue Of Trafficking-Defective Mutant Follicle-Stimulating Hormone Receptors (FSHR) Leading To Premature Ovarian Failure With The Pharmacoperone CAN1405

Abstract Disclosure: A. Ulloa-Aguirre: None. T. Zariñán: None. R. Gutiérrez-Sagal: None. S. Nataraja: None. H.N. Yu: None. E. Jardón-Valadez: None. Inactivating mutations in the follicle-stimulating hormone (FSH) receptor (FSHR) cause premature ovarian failure. Approximately 30% of mutant FSHRs (mutFSHR) are misfolded proteins unable to route to the plasma membrane (PM). We herein tested the effect of a small molecule FSHR ago-allosteric modulator (CAN1405) on the functional rescue of several inactivating mutFSHR. Wild-type (WT) and mutFSHRs A189V, A191I and D224V (at the ectodomain), and four FSHRs with mutations in the serpentine region (SR) of the receptor (D408Y, A419I, I423T, and P519T) were expressed in HEK-293 cells and stimulated with recombinant FSH in the presence of vehicle or CAN1405. Changes in plasma membrane expression and function provoked by CAN1405 exposure were analyzed by immunoblotting and by activation of the cAMP-sensitive pSOMLuc reporter plasmid and ERK1/2 phosphoryation, respectively. Molecular interactions between FSHR and CAN1405, were predicted in silico by computational docking. Exposure of WT and mutFSHR-expressing cells to CAN1405 increased the ratio of mature (PM-expressed) to immature (intracellular) FSHR forms (m/i ratio) of all SR mutant FSHRs (from 0.5 to 3.0) as disclosed by immunoblotting. Similar results were observed when activation of the cAMP-sensitive pSOMLuc reporter and ERK1/2 phosphorylation were examined in FSH-stimulated cells. PM expression of FSHR WT and the activation-defective mutFSHR I423T, but not the FSHRs with mutations in the ectodomain, were also increased by CAN1405. Interesting, in the presence of CAN1405, signaling of the mutFSHR I423T was marginal in terms of activation of the pSOMLuc reporter but not of pERK1/2, suggesting that binding of CAN1405 to the mutFSHR changed the conformation of the receptor allowing functional selectivity on FSH-stimulated signaling. CAN1405 was located in a binding pocket at interhelical upper half of the FSHR. Side chains of residues Y530, A450, T449 S453, L415, H615, M585, V612, I588, and K608 were detected in close interaction with the CAN1045 and the FSHR-CAN1405 complex was likely stabilized by hydrophobic interactions. It is concluded that the small molecule CAN1405 with ago-allosteric properties is an effective pharmacological chaperone that corrects trafficking of SR misfolded mutFSHRs and also increases WT FSHR expression and function. These data demonstrate that allosteric modulators of the FSHR may serve as an effective therapeutic approach for the treatment of infertility in women bearing the WT FSHR or in heterozygous carriers of mutations at the transmembrane domain of this receptor. Presentation: Thursday, June 15, 2023

Novel beta-Glucocerebrosidase Chaperone Compounds Identified from Cell-based Screening Reduce Pathologically Accumulated Glucosylsphingosine in iPS-derived Neuronal Cells.

The beta-glucocerebrosidase (GBA1) gene encodes the lysosomal beta-glucocerebrosidase (GCase) that metabolizes the lipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Biallelic loss-of-function mutations in GBA1 such as L444P cause Gaucher disease (GD), which is the most prevalent lysosomal storage disease and is histopathologically characterized by abnormal accumulation of the GCase substrates GlcCer and GlcSph. GD with neurological symptoms is associated with severe mutations in the GBA1 gene, most of which cause impairment in the process of GCase trafficking to lysosomes. Given that recombinant GCase protein cannot cross the blood-brain barrier due to its high molecular weight, it is invaluable to develop a brain-penetrant small-molecule pharmacological chaperone as a viable therapeutic strategy to boost GCase activity in the central nervous system. Despite considerable efforts to screen potent GCase activators/chaperones, cell-free assays using recombinant GCase protein have yielded compounds with only marginal efficacy and micromolar EC50 that would not have sufficient clinical efficacy or an acceptable safety margin. Therefore, we utilized a fluorescence-labeled GCase suicide inhibitor, MDW933, to directly monitor lysosomal GCase activity and performed a cell-based screening in fibroblasts from a GD patient with homozygotic L444P mutations. Here, we identified novel compounds that increase the fluorescence signal from labeled GCase with L444P mutations in a dose-dependent manner. Secondary assays using an artificial cell-permeable lysosomal GCase substrate also demonstrated that the identified compounds augment lysosomal GCase L444P in the fibroblast. Moreover, those compounds increased the total GCase L444P protein levels, suggesting the pharmacological chaperone-like mechanism of action. To further elucidate the effect of the compounds on the endogenous GCase substrate GlcSph, we generated iPSC-derived dopaminergic neurons with a GBA1 L444P mutation that exhibit GlcSph accumulation invitro. Importantly, the identified compounds reduce GlcSph in iPSC-derived dopaminergic neurons with a GBA1 L444P mutation, indicating that the increase in lysosomal GCase resulting from application of the compounds leads to the clearance of pathologically-accumulated GlcSph. Together, our findings pave the way for developing potent and efficacious GCase chaperone compounds as a potential therapeutic approach for neurological GD.

In Vitro and In Vivo Chaperone Effect of (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one on the C1473G Mutant Tryptophan Hydroxylase 2.

Tryptophan hydroxylase 2 (TPH2) is the key and rate-limiting enzyme of serotonin (5-HT) synthesis in the mammalian brain. The 1473G mutation in the Tph2 gene decreases TPH2 activity in the mouse brain by twofold. (R)-2-amino-6-(1R, 2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is a pharmacological chaperone for aromatic amino acid hydroxylases. In the present study, chaperone effects of BH4 on the mutant C1473G TPH2 were investigated in vitro and in vivo. In vitro BH4 increased the thermal stability (T50 value) of mutant and wild-type TPH2 molecules. At the same time, neither chronic (twice per day for 7 days) intraperitoneal injection of 48.3 mg/kg of BH4 nor a single intraventricular administration of 60 μg of the drug altered the mutant TPH2 activity in the brain of Balb/c mice. This result indicates that although BH4 shows a chaperone effect in vitro, it is unable to increase the activity of mutant TPH2 in vivo.

Elexacaftor/VX-445–mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

Phosphorus Dendrimers for Metal-Free Ligation: Design of Multivalent Pharmacological Chaperones against Gaucher Disease.

The first phosphorus dendrimers built on a cyclotriphosphazene core and decorated with six or twelve monofluorocyclooctyne units were prepared. A simple stirring allowed the grafting of N-hexyl deoxynojirimycin inhitopes onto their surface by copper-free strain promoted alkyne-azide cycloaddition click reaction. The synthesized iminosugars clusters were tested as multivalent inhibitors of the biologically relevant enzymes β-glucocerebrosidase and acid α-glucosidase, involved in Gaucher and Pompe lysosomal storage diseases, respectively. For both enzymes, all the multivalent compounds were more potent than the reference N-hexyl deoxynojirimycin. Remarkably, the final dodecavalent compound proved to be one of the best β-glucocerebrosidase inhibitors described to date. These cyclotriphosphazene-based deoxynojirimycin dendrimers were then evaluated as pharmacological chaperones against Gaucher disease. Not only did these multivalent constructs cross the cell membranes but they were also able to increase β-glucocerebrosidase activity in Gaucher cells. Notably, dodecavalent compound allowed a 1.4-fold enzyme activity enhancement at a concentration as low as 100 nM. These new monofluorocyclooctyne-presenting dendrimers may further find numerous applications in the synthesis of multivalent objects for biological and pharmacological purposes.