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Open Access Publications from the University of California

Scholarly Works (16 results)

  • Article
  • Peer Reviewed
UCLA Previously Published Works (2014)
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 Alzheimer's and Parkinson's diseases.
    Cover page: Disrupting self-assembly and toxicity of amyloidogenic protein oligomers by "molecular tweezers" - from the test tube to animal models.
    • Thesis
    • Peer Reviewed
    UCLA Electronic Theses and Dissertations (2013)

    Aberrant protein self-assembly underlies over 30 human diseases called amyloidoses, for which there are no cures. In these diseases, particular proteins misfold and self-assemble into toxic oligomers that disrupt cellular function, and proceed to form insoluble amyloid fibrils that deposit in specific tissues. A promising strategy for preventing and treating amyloidoses is inhibition or modulation of the self-assembly process to disrupt the formation of the toxic oligomers. In practice, this has proven immensely difficult because the oligomer structures are unknown, are metastable, and do not have distinct binding sites.

    In this dissertation, three primary studies are presented that evaluate and characterize a small molecule, CLR01, which utilizes a novel strategy circumventing these challenges and has been found to be efficacious as an aggregation and toxicity inhibitor in vitro and in vivo. In the first study, CLR01 was evaluated for its ability to rescue synaptic toxicity in cell culture and brain slices. Additionally, it was tested in a transgenic mouse model of AD for its ability to reduce the pathological hallmarks of AD: amyloid plaques and neurofibrillary tangles. This study found positive results in all domains tested; a rescue from amyloid β-protein (Aβ)-induced depletion of synaptic spine density, a rescue of Aβ-induced disruption of basal synaptic transmission and long-term potentiation, and reduction of brain Aβ, hyperphosphorylated tau, and microglia burden. CLR01 also showed low propensity for causing metabolic toxicity or drug-drug interaction, indicating favorable drug-like characteristics.

    In the second study, CLR01's safety and pharmacological profile were characterized in mice. CLR01 was found not to disrupt normal protein assembly, to have a high safety margin in mice, and to penetrate the blood-brain barrier (BBB) at 1-3%. Interestingly, brain levels of CLR01 remained stable for 72 hours following administration despite rapid clearance from the plasma. These results suggest a large safety margin for CLR01 and a pharmacokinetic profile that allows reaching high levels in the brain by administering relatively low doses.

    The third study delineates a detailed optimization of behavioral testing of mice for detection of memory deficits using the Barnes maze, and validates for the first time memory deficits in a triple-transgenic mouse model of AD at the youngest age described in the literature. The study provides a framework for analysis of CLR01's influence on learning and memory deficits in this triple transgenic model. Additionally, the study provides specific and detailed guidelines for optimizing both the performance and the analysis of the Barnes maze in a manner that increases the likelihood of detecting subtle changes in future studies using mouse models of AD. The work described in this dissertation provides a strong foundation supporting formal pre-clinical development of CLR01 as a promising disease-modifying therapeutic drug for AD.

      Cover page: In Vivo Characterization of CLR01, an Aggregation and Toxicity Inhibitor, with an Alzheimer's Disease Focus
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2014)
      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.
        Cover page: Disrupting self-assembly and toxicity of amyloidogenic protein oligomers by molecular tweezers - from the test tube to animal models.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2013)
        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.
          Cover page: Modulators of amyloid protein aggregation and toxicity: EGCG and CLR01
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2014)
          BACKGROUND: The 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 Alzheimers disease and other amyloidoses, here we studied its safety and pharmacokinetics. METHODS: Toxicity 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 Alzheimers disease at 2-m, 12-m, and 22-m of age. Brain levels were measured from 0.5 - 72 h post administration. RESULTS: Examination of CLR01s effect on tubulin polymerization, representing normal protein assembly, showed disruption of the process only when 55-fold excess CLR01 was used, supporting the compounds 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. CONCLUSION: Estimation 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.
            Cover page: Safety and pharmacological characterization of the molecular tweezer CLR01 - a broad-spectrum inhibitor of amyloid proteins toxicity.
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2014)

            Background

            The "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.

            Methods

            Toxicity 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.

            Results

            Examination 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.

            Conclusion

            Estimation 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.
              Cover page: Safety and pharmacological characterization of the molecular tweezer CLR01 – a broad-spectrum inhibitor of amyloid proteins’ toxicity
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2014)
              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.
                Cover page: Molecular tweezers targeting transthyretin amyloidosis.
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2018)
                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.
                  Cover page: Using Molecular Tweezers to Remodel Abnormal Protein Self-Assembly and Inhibit the Toxicity of Amyloidogenic Proteins.
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2013)
                  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.
                    Cover page: A Shortened Barnes Maze Protocol Reveals Memory Deficits at 4-Months of Age in the Triple-Transgenic Mouse Model of Alzheimer's Disease
                    • Article
                    • Peer Reviewed
                    UCLA Previously Published Works (2018)
                    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.
                      Cover page: Using Molecular Tweezers to Remodel Abnormal Protein Self-Assembly and Inhibit the Toxicity of Amyloidogenic Proteins
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