Search

Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies.

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28 days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.

In type-2 diabetes (T2D), islet amyloid polypeptide (IAPP) self-associates into toxic assemblies causing islet β-cell death. Therefore, preventing IAPP toxicity is a promising therapeutic strategy for T2D. The molecular tweezer CLR01 is a supramolecular tool for selective complexation of K residues in (poly)peptides. Surprisingly, it inhibits IAPP aggregation at substoichiometric concentrations even though IAPP has only one K residue at position 1, whereas efficient inhibition of IAPP toxicity requires excess CLR01. The basis for this peculiar behavior is not clear. Here, a combination of biochemical, biophysical, spectroscopic, and computational methods reveals a detailed mechanistic picture of the unique dual inhibition mechanism for CLR01. At low concentrations, CLR01 binds to K1, presumably nucleating nonamyloidogenic, yet toxic, structures, whereas excess CLR01 binds also to R11, leading to nontoxic structures. Encouragingly, the CLR01 concentrations needed for inhibition of IAPP toxicity are safe in vivo, supporting its development toward disease-modifying therapy for T2D.

BackgroundThe "molecular tweezer" CLR01 is a broad-spectrum inhibitor of abnormal protein self-assembly, which acts by binding selectively to Lys residues. CLR01 has been tested in several in vitro and in vivo models of amyloidoses all without signs of toxicity. With the goal of developing CLR01 as a therapeutic drug for Alzheimer's disease and other amyloidoses, here we studied its safety and pharmacokinetics.MethodsToxicity studies were performed in 2-m old wild-type mice. Toxicity was evaluated by serum chemical analysis, histopathology analysis, and qualitative behavioral analysis. Brain penetration studies were performed using radiolabeled CLR01 in both wild-type mice and a transgenic mouse model of Alzheimer's disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration.ResultsExamination of CLR01's effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compound's putative "process-specific" mechanism of action.A single-injection of 100 mg/kg CLR01 in mice - 2,500-fold higher than the efficacious dose reported previously, induced temporary distress and liver injury, but no mortality. Daily injection of doses up to 10 mg/kg did not produce any signs of toxicity, suggesting a high safety margin.The brain penetration of CLR01 was found to be 1 - 3% of blood levels depending on age. Though CLR01 was almost completely removed from the blood by 8 h, unexpectedly, brain levels of CLR01 remained steady over 72 h.ConclusionEstimation of brain levels compared to amyloid β-protein concentrations reported previously suggest that the stoichiometry obtained in vitro and in vivo is similar, supporting the mechanism of action of CLR01.The favorable safety margin of CLR01, together with efficacy shown in multiple animal models, support further development of CLR01 as a disease-modifying agent for amyloidoses.

Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.

Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the molecular tweezers, CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimers disease and Parkinsons disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis.

Despite decades of research, therapy for diseases caused by abnormal protein folding and aggregation (amyloidoses) is limited to treatment of symptoms and provides only temporary and moderate relief to sufferers. The failure in developing successful disease-modifying drugs for amyloidoses stems from the nature of the targets for such drugs - primarily oligomers of amyloidogenic proteins, which are distinct from traditional targets, such as enzymes or receptors. The oligomers are metastable, do not have well-defined structures, and exist in dynamically changing mixtures. Therefore, inhibiting the formation and toxicity of these oligomers likely will require out-of-the-box thinking and novel strategies. We review here the development of a strategy based on targeting the combination of hydrophobic and electrostatic interactions that are key to the assembly and toxicity of amyloidogenic proteins using lysine (K)-specific molecular tweezers (MTs). Our discussion includes a survey of the literature demonstrating the important role of K residues in the assembly and toxicity of amyloidogenic proteins and the development of a lead MT derivative called CLR01, from an inhibitor of protein aggregation in vitro to a drug candidate showing effective amelioration of disease symptoms in animal models of Alzheimers and Parkinsons diseases.

Abnormal protein folding and self-assembly causes over 30 cureless human diseases for which no disease-modifying therapies are available. The common side to all these diseases is formation of aberrant toxic protein oligomers and amyloid fibrils. Both types of assemblies are drug targets, yet each presents major challenges to drug design, discovery, and development. In this review, we focus on two small molecules that inhibit formation of toxic amyloid protein assemblies - the green-tea derivative (-)-epigallocatechin-3-gallate (EGCG), which was identified through a combination of epidemiologic data and a compound library screen, and the molecular tweezer CLR01, whose inhibitory activity was discovered in our group based on rational reasoning, and subsequently confirmed experimentally. Both compounds act in a manner that is not specific to one particular protein and thus are useful against a multitude of amyloidogenic proteins, yet they act via distinct putative mechanisms. CLR01 disrupts protein aggregation through specific binding to lysine residues, whereas the mechanisms underlying the activity of EGCG are only recently beginning to unveil. We discuss current in vitro and, where available, in vivo literature related to EGCG and CLR01's effects on amyloid β-protein, α-synuclein, transthyretin, islet amyloid polypeptide, and calcitonin. We also describe the toxicity, pharmacokinetics, and mechanism of action of each compound. © Versita Sp. z o.o.

Alzheimer's disease is a progressive neurodegenerative disease that manifests as memory loss, cognitive dysfunction, and dementia. Animal models of Alzheimer's disease have been instrumental in understanding the underlying pathological mechanism and in evaluation of potential therapies. The triple transgenic (3 × Tg) mouse model of AD is unique because it recapitulates both pathologic hallmarks of Alzheimer's disease--amyloid plaques and neurofibrillary tangles. The earliest cognitive deficits in this model have been shown at 6-m of age by most groups, necessitating aging of the mice to this age before initiating evaluation of the cognitive effects of therapies. To assess cognitive deficits in the 3 × Tg mice, originally we employed a typical Barnes maze protocol of 15 training trials, but found no significant deficits in aged mice. Therefore, we shortened the protocol to include only 5 training trials to increase difficulty. We found cognitive deficits using this protocol using mainly measures from the probe day, rather than the training trials. This also decreased the effort involved with data analysis. We compared 3 × Tg and wild-type mice at 4-m- and 15-m of age using both the original, long training, and the short training paradigms. We found that differences in learning between 3 × Tg and wild-type mice disappeared after the 4(th) training trial. Measures of learning and memory on the probe day showed significant differences between 3 × Tg and wild-type mice following the short, 5-training trial protocol but not the long, 15-training trial protocol. Importantly, we detected cognitive dysfunction already at 4-m of age in 3 × Tg mice using the short Barnes-maze protocol. The ability to test learning and memory in 4-m old 3 × Tg mice using a shortened Barnes maze protocol offers considerable time and cost savings and provides support for the utilization of this model at pre-pathology stages for therapeutic studies.

The present study was conducted to characterize the effect of ICV benzamil on the time course of DOCA-salt hypertension and the role of the paraventricular nucleus (PVN) in these responses. Uninephrectomized rats were instrumented with a cannula connected to a minipump for infusion of benzamil (BENZ; N=6, 20 nmol/day) or vehicle (VEH; N=6), as well as a transmitter for continuous recording of arterial pressure (AP). DOCA (50 mg) was implanted subcutaneously and AP was recorded for 5 days while rats drank water. Baseline AP was similar (∼100 mmHg) in both groups. Water was replaced with saline and AP recorded for 20 more days. During the first week of saline, group responses were indistinguishable. Starting on Day 7 of saline, marginal group differences in AP emerged. By Day 14, consistent differences emerged, with AP increasing to a greater extent in VEH (∼130 mmHg) than BENZ (∼115 mmHg; p<0.05). On Day 20, AP remained higher in VEH (∼135 mmHg) as compared to BENZ (∼120 mmHg; p <0.01). Brains were then harvested and processed for c-Fos immunohistochemistry in the PVN as an indicator of neuronal activity. Initial results suggest that BENZ reduced PVN c-Fos expression in DOCA-salt rats. We conclude that BENZ treatment attenuates the maintenance phase of DOCA-salt hypertension and that this may be a result of reduced neuronal activity in CNS pressure-regulatory regions, including the PVN. HL 064178 [ABSTRACT FROM AUTHOR]

Background: The molecular tweezer, CLR01, binds to Lys and prevents aggregation of synuclein. Results: CLR01 binds directly to monomeric synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. Conclusion: Aggregation is inhibited by making the protein more diffusive. Significance: The most effective aggregation inhibitors may change monomer dynamics rather than structure. Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examiningα-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 toα-synuclein occurs at the N-terminal Lys-10/Lys- 12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis,α-synuclein ispredominantlymonomeric. The result scan be successfully modeled using a kineticschemein which two aggregation- pronemonomerscanformanencountercomplexthat leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 onα-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins. [ABSTRACT FROM AUTHOR]

Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies.