Molecular Tweezers: Supramolecular Hosts with Broad-Spectrum Biological Applications.

Lysine-selective molecular tweezers (MTs) are supramolecular host molecules displaying a remarkably broad spectrum of biologic activities. MTs act as inhibitors of the self-assembly and toxicity of amyloidogenic proteins using a unique mechanism. They destroy viral membranes and inhibit infection by enveloped viruses, such as HIV-1 and SARS-CoV-2, by mechanisms unrelated to their action on protein self-assembly. They also disrupt biofilm of Gram-positive bacteria. The efficacy and safety of MTs have been demonstrated in vitro, in cell culture, and in vivo, suggesting that these versatile compounds are attractive therapeutic candidates for various diseases, infections, and injuries. A lead compound called CLR01 has been shown to inhibit the aggregation of various amyloidogenic proteins, facilitate their clearance in vivo, prevent infection by multiple viruses, display potent anti-biofilm activity, and have a high safety margin in animal models. The inhibitory effect of CLR01 against amyloidogenic proteins is highly specific to abnormal self-assembly of amyloidogenic proteins with no disruption of normal mammalian biologic processes at the doses needed for inhibition. Therapeutic effects of CLR01 have been demonstrated in animal models of proteinopathies, lysosomal-storage diseases, and spinal-cord injury. Here we review the activity and mechanisms of action of these intriguing compounds and discuss future research directions. SIGNIFICANCE STATEMENT: Molecular tweezers are supramolecular host molecules with broad biological applications, including inhibition of abnormal protein aggregation, facilitation of lysosomal clearance of toxic aggregates, disruption of viral membranes, and interference of biofilm formation by Gram-positive bacteria. This review discusses the molecular and cellular mechanisms of action of the molecular tweezers, including the discovery of distinct mechanisms acting in vitro and in vivo, and the application of these compounds in multiple preclinical disease models.

Anti-amyloid treatment is broadly effective in neuronopathic mucopolysaccharidoses and synergizes with gene therapy in MPS-IIIA.

Mucopolysaccharidoses (MPSs) are childhood diseases caused by inherited deficiencies in glycosaminoglycan degradation. Most MPSs involve neurodegeneration, which to date is untreatable. Currently, most therapeutic strategies aim at correcting the primary genetic defect. Among these strategies, gene therapy has shown great potential, although its clinical application is challenging. We have shown previously in an MPS-IIIA mouse model that the molecular tweezer (MT) CLR01, a potent, broad-spectrum anti-amyloid small molecule, inhibits secondary amyloid storage, facilitates amyloid clearance, and protects against neurodegeneration. Here, we demonstrate that combining CLR01 with adeno-associated virus (AAV)-mediated gene therapy, targeting both the primary and secondary pathologic storage in MPS-IIIA mice, results in a synergistic effect that improves multiple therapeutic outcomes compared to each monotherapy. Moreover, we demonstrate that CLR01 is effective therapeutically in mouse models of other forms of neuronopathic MPS, MPS-I, and MPS-IIIC. These strongly support developing MTs as an effective treatment option for neuronopathic MPSs, both on their own and in combination with gene therapy, to improve therapeutic efficacy and translation into clinical application.

ARL6IP5 Ameliorates α-Synuclein Burden by Inducing Autophagy via Preventing Ubiquitination and Degradation of ATG12.

Recent advanced studies in neurodegenerative diseases have revealed several links connecting autophagy and neurodegeneration. Autophagy is the major cellular degradation process for the removal of toxic protein aggregates responsible for neurodegenerative diseases. More than 30 autophagy-related proteins have been identified as directly participating in the autophagy process. Proteins regulating the process of autophagy are much more numerous and unknown. To address this, in our present study, we identified a novel regulator (ARL6IP5) of neuronal autophagy and showed that the level of ARL6IP5 decreases in the brain with age and in Parkinson's disease in mice and humans. Moreover, a cellular model of PD (Wild type and A53T mutant α-synuclein overexpression) has also shown decreased levels of ARL6IP5. ARL6IP5 overexpression reduces α-synuclein aggregate burden and improves cell survival in an A53T model of Parkinson's disease. Interestingly, detailed mechanistic studies revealed that ARL6IP5 is an autophagy inducer. ARL6IP5 enhances Rab1-dependent autophagosome initiation and elongation by stabilizing free ATG12. We report for the first time that α-synuclein downregulates ARL6IP5 to inhibit autophagy-dependent clearance of toxic aggregates that exacerbate neurodegeneration.

mTOR Inhibition with Sirolimus in Multiple System Atrophy: A Randomized, Double-Blind, Placebo-Controlled Futility Trial and 1-Year Biomarker Longitudinal Analysis.

BACKGROUND: Multiple system atrophy (MSA) is a fatal neurodegenerative disease characterized by the aggregation of α-synuclein in glia and neurons. Sirolimus (rapamycin) is an mTOR inhibitor that promotes α-synuclein autophagy and reduces its associated neurotoxicity in preclinical models. OBJECTIVE: To investigate the efficacy and safety of sirolimus in patients with MSA using a futility design. We also analyzed 1-year biomarker trajectories in the trial participants. METHODS: Randomized, double-blind, parallel group, placebo-controlled clinical trial at the New York University of patients with probable MSA randomly assigned (3:1) to sirolimus (2-6 mg daily) for 48 weeks or placebo. Primary endpoint was change in the Unified MSA Rating Scale (UMSARS) total score from baseline to 48 weeks. (ClinicalTrials.gov NCT03589976). RESULTS: The trial was stopped after a pre-planned interim analysis met futility criteria. Between August 15, 2018 and November 15, 2020, 54 participants were screened, and 47 enrolled and randomly assigned (35 sirolimus, 12 placebo). Of those randomized, 34 were included in the intention-to-treat analysis. There was no difference in change from baseline to week 48 between the sirolimus and placebo in UMSARS total score (mean difference, 2.66; 95% CI, -7.35-6.91; P = 0.648). There was no difference in UMSARS-1 and UMSARS-2 scores either. UMSARS scores changes were similar to those reported in natural history studies. Neuroimaging and blood biomarker results were similar in the sirolimus and placebo groups. Adverse events were more frequent with sirolimus. Analysis of 1-year biomarker trajectories in all participants showed that increases in blood neurofilament light chain (NfL) and reductions in whole brain volume correlated best with UMSARS progression. CONCLUSIONS: Sirolimus for 48 weeks was futile to slow the progression of MSA and had no effect on biomarkers compared to placebo. One-year change in blood NfL and whole brain atrophy are promising biomarkers of disease progression for future clinical trials. © 2022 International Parkinson and Movement Disorder Society.

Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS129-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS129-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

Lysine-selective molecular tweezers are cell penetrant and concentrate in lysosomes.

Lysine-selective molecular tweezers are promising drug candidates against proteinopathies, viral infection, and bacterial biofilm. Despite demonstration of their efficacy in multiple cellular and animal models, important questions regarding their mechanism of action, including cell penetrance and intracellular distribution, have not been answered to date. The main impediment to answering these questions has been the low intrinsic fluorescence of the main compound tested to date, called CLR01. Here, we address these questions using new fluorescently labeled molecular tweezers derivatives. We show that these compounds are internalized in neurons and astrocytes, at least partially through dynamin-dependent endocytosis. In addition, we demonstrate that the molecular tweezers concentrate rapidly in acidic compartments, primarily lysosomes. Accumulation of molecular tweezers in lysosomes may occur both through the endosomal-lysosomal pathway and via the autophagy-lysosome pathway. Moreover, by visualizing colocalization of molecular tweezers, lysosomes, and tau aggregates we show that lysosomes likely are the main site for the intracellular anti-amyloid activity of molecular tweezers. These findings have important implications for the mechanism of action of molecular tweezers in vivo, explaining how administration of low doses of the compounds achieves high effective concentrations where they are needed, and supporting the development of these compounds as drugs for currently cureless proteinopathies.

The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301S-tau transgenic mice.

BACKGROUND: Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein aggregation. A lead MT, called CLR01, has been demonstrated to inhibit the aggregation and toxicity of multiple amyloidogenic proteins in vitro and in vivo. Previously, we evaluated the effect of CLR01 in the 3 × Tg mouse model of Alzheimer's disease, which overexpresses mutant human presenilin 1, amyloid ß-protein precursor, and tau and found that subcutaneous administration of the compound for 1 month led to a robust reduction of amyloid plaques, neurofibrillary tangles, and microgliosis. CLR01 also has been demonstrated to inhibit tau aggregation in vitro and tau seeding in cell culture, yet because in Alzheimer's disease (AD) and in the 3 × Tg model, tau hyperphosphorylation and aggregation are thought to be downstream of Aß insults, the study in this model left open the question whether CLR01 affected tau in vivo directly or indirectly. METHODS: To determine if CLR01 could ameliorate tau pathology directly in vivo, we tested the compound similarly using the P301S-tau (line PS19) mouse model. Mice were administered 0.3 or 1.0 mg/kg per day CLR01 and tested for muscle strength and behavioral deficits, including anxiety- and disinhibition-like behavior. Their brains then were analyzed by immunohistochemical and biochemical assays for pathological forms of tau, neurodegeneration, and glial pathology. RESULTS: CLR01 treatment ameliorated muscle-strength deterioration, anxiety-, and disinhibition-like behavior. Improved phenotype was associated with decreased levels of pathologic tau forms, suggesting that CLR01 exerts a direct effect on tau in vivo. Limitations of the study included a relatively short treatment period of the mice at an age in which full pathology is not yet developed. In addition, high variability in this model lowered the statistical significance of the findings of some outcome measures. CONCLUSIONS: The findings suggest that CLR01 is a particularly attractive candidate for the treatment of AD because it targets simultaneously the two major pathogenic proteins instigating and propagating the disease, amyloid ß-protein (Aß), and tau, respectively. In addition, our study suggests that CLR01 can be used for the treatment of other tauopathies in the absence of amyloid pathology.

An insight into the inhibition of fibrillation process verses disaggregation of preformed fibrils of bovine serum albumin by isoprenaline hydrochloride.

This study is based on the analysis of the recent trend of medication in neurodegenerative diseases. Due to the asymptomatic nature of the diseases, medication delays. Therefore, mechanism of medication assists in removal of the symptoms. Therefore, in order to find out remedy for complete prevention of the disease we have considered "inhibition verses disaggregation" study. Various biophysical techniques such as turbidity measurement (TM), Thioflavin T (ThT) binding assays, circular dichroism (CD), transmission electron microscopy (TEM) etc. has been performed. Isoprenaline hydrochloride (ISO) was a good candidate for inhibition and disaggregation of preformed fibrils of BSA. Therefore, it is concluded that inhibition of fibrillation process was more momentous, effective procedure in restricting the aggregation by stabilizing the native conformation of BSA than the removal of preformed amyloid fibrils under in vitro condition. Forwarding ahead, to understand the efficiency of the two processes under in vivo condition, this study can be applied on animal models so that we can look forward on human beings as well for the development of vaccines. This study is concerned about the applied aspect of research in future so that we can hope for prevention of the disease instead of only removal of the symptoms.

Different Inhibitors of Aß42-Induced Toxicity Have Distinct Metal-Ion Dependency.

Oligomers of amyloid ß-protein (Aß) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of Aß has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn2+ can strongly modulate the aggregation of Aß in vitro and both promote and inhibit the neurotoxicity of Aß, depending on the experimental conditions. Thus, successful inhibitors of Aß self-assembly may have to inhibit the toxicity not only of Aß oligomers themselves but also of Aß-Zn2+ complexes. However, there has been relatively little research investigating the effects of Aß self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of Aß42 C-terminal fragments (CTFs), some of which have been shown to inhibit Aß oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of Aß42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, ß-sheet, and fibril formation by Aß42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with Aß42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to Aß42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both Aß42 oligomerization and Aß42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of Aß42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of Aß42 alone. In agreement with these results, CLR01 inhibited ß-sheet and fibril formation in Aß42-Zn2+ complexes. Our data suggest that, for the development of efficient therapeutic agents, inhibitors of Aß self-assembly and toxicity should be examined in the presence of relevant metal ions and that molecular tweezers may be particularly attractive candidates for therapy development.

Prion protein transcription is auto-regulated through dynamic interactions with G-quadruplex motifs in its own promoter.

Cellular prion protein (PrP) misfolds into an aberrant and infectious scrapie form (PrPSc) that lead to fatal transmissible spongiform encephalopathies (TSEs). Association of prions with G-quadruplex (GQ) forming nucleic acid motifs has been reported, but implications of these interactions remain elusive. Herein, we show that the promoter region of the human prion gene (PRNP) contains two putative GQ motifs (Q1 and Q2) that assume stable, hybrid, intra-molecular quadruplex structures and bind with high affinity to PrP. Here, we investigate the ability of PrP to bind to the quadruplexes in its own promoter. We used a battery of techniques including SPR, NMR, CD, MD simulations and cell culture-based reporter assays. Our results show that PrP auto-regulates its expression by binding and resolving the GQs present in its own promoter. Furthermore, we map this resolvase-like activity to the N-terminal region (residues 23-89) of PrP. Our findings highlight a positive transcriptional-translational feedback regulation of the PRNP gene by PrP through dynamic unwinding of GQs in its promoter. Taken together, our results shed light on a yet unknown mechanism of regulation of the PRNP gene. This work provides the necessary framework for a plethora of studies on understanding the regulation of PrP levels and its implications in prion pathogenesis.

CLR01 protects dopaminergic neurons in vitro and in mouse models of Parkinson's disease.

Parkinson's disease (PD) affects millions of patients worldwide and is characterized by alpha-synuclein aggregation in dopamine neurons. Molecular tweezers have shown high potential as anti-aggregation agents targeting positively charged residues of proteins undergoing amyloidogenic processes. Here we report that the molecular tweezer CLR01 decreased aggregation and toxicity in induced pluripotent stem cell-derived dopaminergic cultures treated with PD brain protein extracts. In microfluidic devices CLR01 reduced alpha-synuclein aggregation in cell somas when axonal terminals were exposed to alpha-synuclein oligomers. We then tested CLR01 in vivo in a humanized alpha-synuclein overexpressing mouse model; mice treated at 12 months of age when motor defects are mild exhibited an improvement in motor defects and a decreased oligomeric alpha-synuclein burden. Finally, CLR01 reduced alpha-synuclein-associated pathology in mice injected with alpha-synuclein aggregates into the striatum or substantia nigra. Taken together, these results highlight CLR01 as a disease-modifying therapy for PD and support further clinical investigation.

A multiparametric analysis of the synergistic impact of anti-Parkinson's drugs on the fibrillation of human serum albumin.

Protein aggregation have been associated with several human neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases. There are several small molecules that can reduce aggregation of proteins. The present study aimed to test the hypothesis that the application of more than one inhibitor either simultaneously or consecutively may result in more efficient inhibition of protein aggregation. To this end, the anti-amyloidogenic behaviour of benserazide hydrochloride (BH) and levodopa (LD) individually and in combination (BH + LD) was investigated using various biophysical, microscopic, and computational techniques. BH, LD, and BH + LD treatments showed inhibitory effects on protein aggregation and had the ability to minimise the amyloid-induced cytotoxicity in human neuroblastoma cell line (SH-SY5Y). The two drugs in combination showed synergism (combination index, CI < 1) between them. These drugs also destabilised the preformed fibrils of human serum albumin (HSA). Our studies consistently showed that the BH + LD treatment showed highest efficacy towards inhibition and disaggregation of amyloid fibrils in comparison to treatment with BH and LD individually. Therefore, application of drugs in combination against fibrillogenesis may represent a new route for development of means for prevention or delaying of the aggregation-related diseases.

A routinely used protein staining dye acts as an inhibitor of wild type and mutant alpha-synuclein aggregation and modulator of neurotoxicity.

Inhibition of amyloid formation along with modulation of toxicity employing small molecules is emerging as a potential therapeutic approach for protein misfolding disorders which includes Parkinson's disease, Alzheimer's disease and Multiple System Atrophy etc. Countless current interventional strategies for treating α-synucleinopathies consider using peptidic and non-peptidic inhibitors for arresting fibrillisation, disrupting existing fibrils and reducing associated toxicity. One group of molecules less exploited in this regard are triphenylmethane dyes. Herein we tested the inhibitory effect of two routinely used protein staining dyes viz Coomassie Brilliant blue G (CBBG) and Coomassie Brilliant blue R (CBBR) employing several biophysical and cell based methods. Our results showed that both the dyes not only efficiently inhibit fibrillisation but also disrupt existing fibrils. Nonetheless, only CBBR prevented the appearance of A11 epitopes which are marker of toxicity. Moreover, CBBR was also able to stall fibrillisation of A53T mutant α-synuclein and reduce associated neurotoxicity. This study thus reports the potential of CBBR as a therapeutic molecule.

Inhibitory growth evaluation and apoptosis induction in MCF-7 cancer cells by new 5-aryl-2-butylthio-1,3,4-oxadiazole derivatives.

BACKGROUND: Cancer has become one of the global health issues and it is the life-threatening disease characterized by unrestrained growth of cells. Despite various advances being adopted by chemotherapeutic management, the use of the current anticancer drugs such as Doxorubicin, Asparginase, Methotrexate, Vincristine remains limited due to high toxicity, side effects and developing drug resistance. Apoptosis is a crucial cellular process and improper regulation of apoptotic signaling pathways may lead to cancer formation. Subsequently, the synthesis of effective chemotherapeutic agents that can induce apoptosis in tumor cell has emerged as a significant approach in cancer drug discovery. METHODS: The goal of this work is to develop a potential antitumor agent exerting significant inhibitory effects on cancer cell and low cytotoxicity, for which we focused on the structural features of 1,3,4-oxadiazoles as it a privileged scaffold in modern medicinal chemistry and have the ability to inhibit growth factors, enzymes and kinases potentially involved in the attainment of cellular immortality and carcinogenesis. RESULT: In vitro MTT screening assay showed the compound 5-aminophenyl-2-butylthio-1,3,4-oxadiazole (5e) showing the highest inhibitory effect against MCF-7 cancer cell with IC50 value 10.05 ± 1.08 µM while it is much safer and less toxic on normal cell line (HEK-293). The dose-dependent treatment of MCF-7 cells with 5e resulted in inhibition of cell migration in the wound healing assay. The flow-cytometry analysis showed the cells arrested in G0/G1 phase of the cell cycle. Compound 5e induced apoptosis of MCF-7 cells was characterized using DAPI staining and Annexin V-PE/7-AAD dual binding assay. Reduction of NBT by compound 5e showed a reduced generation of ROS. Western blotting studies showed high activation of apoptotic protein Caspase3 and decrease in expression of anti-apoptotic protein BCL-2. CONCLUSION: Based on the results of in vitro studies, it could be concluded that compound 5e showed a significant inhibitory growth effect on MCF-7 cells and have the potential to be developed as lead molecule and further structural modifications may result in promising new anticancer agents.

Anti-Parkinsonian L-Dopa can also act as anti-systemic amyloidosis-A mechanistic exploration.

In spite of the fact that amyloid related neurodegenerative illnesses and non-neuropathic systemic amyloidosis have allured the research endeavors, as no cure has been announced yet apart from symptomatic treatment. Therapeutic agents which can reduce or disaggregate those toxic oligomers and fibrillar species have been studied with more compounds are on their way. The current research work describes comprehensive biophysical, computational and microscopic studies which reveal that L-3, 4-dihydroxyphenylalanine (L-Dopa) have indisputable efficacy to hinder the heat induced amyloid fibrillation of the human lysozyme (HL) and also preserve the fibril disaggregating potential. The IC50 value of L-Dopa is calculated to be 63.0±0.09µM. L-Dopa intervenes in the process of amyloid fibrillogenesis through hydrophobic interaction and hydrogen bond formation with the amino acid residues found in the amyloid fibril forming prone region of HL as clarified by molecular simulation data. L-Dopa also disaggregates the mature amyloid fibrils into some unorganized species and the DC50 value was estimated to be 19.95±0.063µM. Hence, L-Dopa and related compounds can act as effective inhibitors in the therapeutic development to combat systemic amyloidosis.

Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight.

Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer's dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aß-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils.