Search

Protein misfolding and aggregation are hallmarks of many severe neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease. As a supramolecular ligand that binds to lysine and arginine residues, the molecular tweezer CLR01 was found to modify the aggregation pathway of disease-relevant proteins in vitro and in vivo with beneficial effects on toxicity. However, the molecular mechanisms of how tweezers exert these effects remain mainly unknown, hampering further drug development. Here, we investigate the modulation mechanism of unfolding and aggregation pathways of SOD1, which are involved in amyotrophic lateral sclerosis (ALS), by CLR01. Using a truncated version of the wildtype SOD1 protein, SOD1bar , we show that CLR01 acts on the first step of the aggregation pathway, the unfolding of the SOD1 monomer. CLR01 increases, by ∼10 °C, the melting temperatures of the A4V and G41D SOD1 mutants, which are commonly observed mutations in familial ALS. Molecular dynamics simulations and binding free energy calculations as well as native mass spectrometry and mutational studies allowed us to identify K61 and K92 as binding sites for the tweezers to mediate the stability increase. The data suggest that the modulation of SOD1 conformational stability is a promising target for future developments of supramolecular ligands against neurodegenerative diseases.

Using supramolecules for protein function regulation is an effective strategy in chemical biology and drug discovery. However, due to the presence of multiple binding sites on protein surfaces, protein function regulation via selective binding of supramolecules is challenging. Recently, the functions of 14-3-3 proteins, which play an important role in regulating intracellular signaling pathways via protein–protein interactions, have been modulated using a supramolecular tweezer, CLR01. However, the binding mechanisms of the tweezer molecule to 14-3-3 proteins are still unclear, which has hindered the development of novel supramolecules targeting the 14-3-3 proteins. Herein, the binding mechanisms of the tweezer to the lysine residues on 14-3-3σ (an isoform in 14-3-3 protein family) were explored by well-tempered metadynamics. The results indicated that the inclusion complex formed between the protein and supramolecule is affected by both kinetic and thermodynamic factors. In particular, simulations confirmed that K214 could form a strong binding complex with the tweezer; the binding free energy was calculated to be −10.5 kcal·mol−1 with an association barrier height of 3.7 kcal·mol−1. In addition, several other lysine residues on 14-3-3σ were identified as being well-recognized by the tweezer, which agrees with experimental results, although only K214/tweezer was co-crystallized. Additionally, the binding mechanisms of the tweezer to all lysine residues were analyzed by exploring the representative conformations during the formation of the inclusion complex. This could be helpful for the development of new inhibitors based on tweezers with more functions against 14-3-3 proteins via modifications of CLR01. We also believe that the proposed computational strategies can be extended to understand the binding mechanism of multi-binding sites proteins with supramolecules and will, thus, be useful toward drug design.

Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the "molecular tweezer" inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury.

Human cytomegalovirus (HCMV) uses two major ways for virus dissemination: infection by cell-free virus and direct cell-to-cell spread. Neutralizing antibodies can efficiently inhibit infection by cell-free virus but mostly fail to prevent cell-to-cell transmission. Here, we show that the ‘molecular tweezer’ CLR01, a broad-spectrum antiviral agent, is not only highly active against infection with cell-free virus but most remarkably inhibits antibody-resistant direct cell-to-cell spread of HCMV. The inhibition of cell-to-cell spread by CLR01 was not limited to HCMV but was also shown for the alphaherpesviruses herpes simplex viruses 1 and 2 (HSV-1, -2). CLR01 is a rapid acting small molecule that inhibits HCMV entry at the attachment and penetration steps. Electron microscopy of extracellular virus particles indicated damage of the viral envelope by CLR01, which likely impairs the infectivity of virus particles. The rapid inactivation of viral particles by CLR01, the viral envelope as the main target, and the inhibition of virus entry at different stages are presumably the key to inhibition of cell-free virus infection and cell-to-cell spread by CLR01. Importance: While cell-free spread enables the human cytomegalovirus (HCMV) and other herpesviruses to transmit between hosts, direct cell-to-cell spread is thought to be more relevant for in vivo dissemination within infected tissues. Cell-to-cell spread is resistant to neutralizing antibodies, thus contributing to the maintenance of virus infection and virus dissemination in the presence of an intact immune system. Therefore, it would be therapeutically interesting to target this mode of spread in order to treat severe HCMV infections and to prevent dissemination of virus within the infected host. The molecular tweezer CLR01 exhibits broad-spectrum antiviral activity against a number of enveloped viruses and efficiently blocks antibody-resistant cell-to-cell spread of HCMV, thus representing a novel class of small molecules with promising antiviral activity.

The supramolecular complex formed between protein and designed molecule has become one of the most efficient ways to modify protein functions. As one of the more well-studied model systems, 14-3-3 family proteins play an important role in regulating intracellular signaling pathways via protein-protein interactions. In this work, we selected 14-3-3σ as the target protein. Molecular dynamics simulations and binding free energy calculations were applied to identify the possible binding sites and understand its recognition ability of the supramolecular inhibitor, the tweezer molecule (CLR01). On the basis of our simulation, major interactions between lysine residues and CLR01 come from the van der Waals interactions between the long alkyl chain of lysine and the cavity formed by the norbornadiene and benzene rings of the inhibitor. Apart from K214, which was found to be crystallized with this inhibitor, other lysine sites have also shown their abilities to form inclusion complexes with the inhibitor. Such non-specific recognition features of CLR01 against 14-3-3σ can be used in the modification of protein functions via supramolecular chemistry.

Spinal cord injury causes neuronal death, limiting subsequent regeneration and recovery. Thus, there is a need to develop strategies for improving neuronal survival after injury. Relative to our understanding of axon regeneration, comparatively little is known about the mechanisms that promote the survival of damaged neurons. To address this, we took advantage of lamprey giant reticulospinal neurons whose large size permits detailed examination of post-injury molecular responses at the level of individual, identified cells. We report here that spinal cord injury caused a select subset of giant reticulospinal neurons to accumulate synuclein, a synaptic vesicle-associated protein best known for its atypical aggregation and causal role in neurodegeneration in Parkinson's and other diseases. Post-injury synuclein accumulation took the form of punctate aggregates throughout the somata and occurred selectively in dying neurons, but not in those that survived. In contrast, another synaptic vesicle protein, synaptotagmin, did not accumulate in response to injury. We further show that the post-injury synuclein accumulation was greatly attenuated after single dose application of either the “molecular tweezer” inhibitor, CLR01, or a translation-blocking synuclein morpholino. Consequently, reduction of synuclein accumulation not only improved neuronal survival, but also increased the number of axons in the spinal cord proximal and distal to the lesion. This study is the first to reveal that reducing synuclein accumulation is a novel strategy for improving neuronal survival after spinal cord injury. [ABSTRACT FROM AUTHOR]

Using supramolecules for protein function regulation is an effective strategy in chemical biology and drug discovery. However, due to the presence of multiple binding sites on protein surfaces, protein function regulation via selective binding of supramolecules is challenging. Recently, the functions of 14-3-3 proteins, which play an important role in regulating intracellular signaling pathways via protein-protein interactions, have been modulated using a supramolecular tweezer, CLR01. However, the binding mechanisms of the tweezer molecule to 14-3-3 proteins are still unclear, which has hindered the development of novel supramolecules targeting the 14-3-3 proteins. Herein, the binding mechanisms of the tweezer to the lysine residues on 14-3-3σ (an isoform in 14-3-3 protein family) were explored by well-tempered metadynamics. The results indicated that the inclusion complex formed between the protein and supramolecule is affected by both kinetic and thermodynamic factors. In particular, simulations confirmed that K214 could form a strong binding complex with the tweezer; the binding free energy was calculated to be -10.5 kcal·mol -1 with an association barrier height of 3.7 kcal·mol -1 . In addition, several other lysine residues on 14-3-3σ were identified as being well-recognized by the tweezer, which agrees with experimental results, although only K214/tweezer was co-crystallized. Additionally, the binding mechanisms of the tweezer to all lysine residues were analyzed by exploring the representative conformations during the formation of the inclusion complex. This could be helpful for the development of new inhibitors based on tweezers with more functions against 14-3-3 proteins via modifications of CLR01. We also believe that the proposed computational strategies can be extended to understand the binding mechanism of multi-binding sites proteins with supramolecules and will, thus, be useful toward drug design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhou, Shi, Wang and Xu.)

The protein α-synuclein (α-syn) self-assembles under abnormal conditions into toxic aggregates thought to be a central cause of pathology in neurodegenerative diseases such as Parkinson's Disease (PD). A promising approach for treating PD is to inhibit the abnormal selfassembly of α-syn in the brain by using small molecules called "molecular tweezers" that the Bitan laboratory has been developing. Molecular tweezers bind to lysine (Lys) residues and prevent both hydrophobic and electrostatic interactions that are key to abnormal self-assembly. The molecular tweezer CLR01 inhibits the self-assembly of α-syn in vitro and in vivo by preferentially binding to Lys at positions 10 and/or 12 and at the region spanning residues 43-58. This leads me to investigate how effective CLR01 is in preventing aggregation when the amino acid at the binding site is substituted, by using Lys to Ala variants of α-syn at positions 43 and 58. I hypothesize that CLR01 will prevent more effectively aggregation in the wild type (WT) than it will in the two variants because essential binding sites will be missing in these variants. As my main experimental method, I use the Thioflavin-T fluorescence assay to measure the amount and kinetics of -pleated sheet formation, which is analogous to α-syn aggregation regardless of the absence or concentration of CLR01. This study will provide insight into the preferred binding site of CLR01 and the behavior of α-syn containing amino acid substitutions and thus will increase our understanding of this important mechanism underlying PD and potentially direct future drug development efforts. [ABSTRACT FROM AUTHOR]

The supramolecular complex formed between protein and designed molecule has become one of the most efficient ways to modify protein functions. As one of the more well-studied model systems, 14-3-3 family proteins play an important role in regulating intracellular signaling pathways via protein-protein interactions. In this work, we selected 14-3-3σ as the target protein. Molecular dynamics simulations and binding free energy calculations were applied to identify the possible binding sites and understand its recognition ability of the supramolecular inhibitor, the tweezer molecule (CLR01). On the basis of our simulation, major interactions between lysine residues and CLR01 come from the van der Waals interactions between the long alkyl chain of lysine and the cavity formed by the norbornadiene and benzene rings of the inhibitor. Apart from K214, which was found to be crystallized with this inhibitor, other lysine sites have also shown their abilities to form inclusion complexes with the inhibitor. Such non-specific recognition features of CLR01 against 14-3-3σ can be used in the modification of protein functions via supramolecular chemistry.