Noncovalent, Electrostatic Interactions Induce Positively Cooperative Binding of Small Molecules to Alzheimer's and Parkinson's Disease-Related Amyloids.

Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases

Enzyme-Linked Immunosorbent Assay-Based Method to Quantify the Association of Small Molecules with Aggregated Amyloid Peptides

This paper describes a simple enzyme linked immunosorbent assay (ELISA) protocol for quantifying the binding of small molecules to aggregated β-amyloid (Aβ) peptides. Amyloid-targeting small molecules have attracted wide interest as potential agents for the treatment or diagnosis of neurodegenerative disorders such as Alzheimer’s disease. The lack of general methods to evaluate small molecule–amyloid binding interactions, however, has significantly limited the number of amyloid-targeting molecules that have been studied to date. Here, we demonstrate a general method to quantify small molecule–amyloid binding interactions via a modified quantitative ELISA protocol. A key feature of this protocol is the treatment of commercial ELISA plates with an air plasma to help maintain the desired β-sheet content of the aggregated Aβ upon immobilization of these peptides on to the polystyrene surface. We developed an ELISA-based competition assay on these air plasma-treated plates and evaluated the binding of five previously known amyloid-binding small molecules to aggregated Aβ. We show that this general ELISA-based competition assay can be used to quantify small molecule–amyloid binding interactions in the low nanomolar to low micromolar range, which is the typical range of affinities for many amyloid-targeting diagnostic agents under current development. This simple protocol for quantifying the interaction of small molecules with aggregated Aβ peptides overcomes many limitations of previously reported spectroscopic or radioactivity assays and may, therefore, facilitate the screening and evaluation of a more structurally diverse set of amyloid-targeting agents than had previously been possible

Noncovalent, Electrostatic Interactions Induce Positively Cooperative Binding of Small Molecules to Alzheimer's and Parkinson's Disease-Related Amyloids.

Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases

Oligovalent Amyloid-Binding Agents Reduce SEVI-Mediated Enhancement of HIV-1 Infection

This paper evaluates the use of oligovalent amyloid-binding molecules as potential agents that can reduce the enhancement of human immunodeficiency virus-1 (HIV-1) infection in cells by semen-derived enhancer of virus infection (SEVI) fibrils. These naturally occurring amyloid fibrils found in semen have been implicated as mediators that can facilitate the attachment and internalization of HIV-1 virions to immune cells. Molecules that are capable of reducing the role of SEVI in HIV-1 infection may, therefore, represent a novel strategy to reduce the rate of sexual transmission of HIV-1 in humans. Here, we evaluated a set of synthetic, oligovalent derivatives of benzothiazole aniline (BTA, a known amyloid-binding molecule) for their capability to bind cooperatively to aggregated amyloid peptides and to neutralize the effects of SEVI in HIV-1 infection. We demonstrate that these BTA derivatives exhibit a general trend of increased binding to aggregated amyloids as a function of increasing valence number of the oligomer. Importantly, we find that oligomers of BTA show improved capability to reduce SEVI-mediated infection of HIV-1 in cells compared to a BTA monomer, with the pentamer exhibiting a 65-fold improvement in efficacy compared to a previously reported monomeric BTA derivative. These results, thus, support the use of amyloid-targeting molecules as potential supplements for microbicides to curb the spread of HIV-1 through sexual contact

The small molecule CA140 inhibits the neuroinflammatory response in wild-type mice and a mouse model of AD

Abstract Background Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer’s disease (AD). Thus, modulating the neuroinflammatory response represents a potential therapeutic strategy for treating neurodegenerative diseases. Several recent studies have shown that dopamine (DA) and its receptors are expressed in immune cells and are involved in the neuroinflammatory response. Thus, we recently developed and synthesized a non-self-polymerizing analog of DA (CA140) and examined the effect of CA140 on neuroinflammation. Methods To determine the effects of CA140 on the neuroinflammatory response, BV2 microglial cells were pretreated with lipopolysaccharide (LPS, 1 μg/mL), followed by treatment with CA140 (10 μM) and analysis by reverse transcription-polymerase chain reaction (RT-PCR). To examine whether CA140 alters the neuroinflammatory response in vivo, wild-type mice were injected with both LPS (10 mg/kg, intraperitoneally (i.p.)) and CA140 (30 mg/kg, i.p.), and immunohistochemistry was performed. In addition, familial AD (5xFAD) mice were injected with CA140 or vehicle daily for 2 weeks and examined for microglial and astrocyte activation. Results Pre- or post-treatment with CA140 differentially regulated proinflammatory responses in LPS-stimulated microglia and astrocytes. Interestingly, CA140 regulated D1R levels to alter LPS-induced proinflammatory responses. CA140 significantly downregulated LPS-induced phosphorylation of ERK and STAT3 in BV2 microglia cells. In addition, CA140-injected wild-type mice exhibited significantly decreased LPS-induced microglial and astrocyte activation. Moreover, CA140-injected 5xFAD mice exhibited significantly reduced microglial and astrocyte activation. Conclusions CA140 may be beneficial for preventing and treating neuroinflammatory-related diseases, including AD

A Tetra(Ethylene Glycol) Derivative of Benzothiazole Aniline Enhances Ras-Mediated Spinogenesis

The tetra(ethylene glycol) derivative of benzothiazole aniline, BTA-EG(4), is a novel amyloid-binding small molecule that can penetrate the blood–brain barrier and protect cells from Aβ-induced toxicity. However, the effects of Aβ-targeting molecules on other cellular processes, including those that modulate synaptic plasticity, remain unknown. We report here that BTA-EG(4) decreases Aβ levels, alters cell surface expression of amyloid precursor protein (APP), and improves memory in wild-type mice. Interestingly, the BTA-EG(4)-mediated behavioral improvement is not correlated with LTP, but with increased spinogenesis. The higher dendritic spine density reflects an increase in the number of functional synapses as determined by increased miniature EPSC (mEPSC) frequency without changes in presynaptic parameters or postsynaptic mEPSC amplitude. Additionally, BTA-EG(4) requires APP to regulate dendritic spine density through a Ras signaling-dependent mechanism. Thus, BTA-EG(4) may provide broad therapeutic benefits for improving neuronal and cognitive function, and may have implications in neurodegenerative disease therapy

Hexa (ethylene glycol) derivative of benzothiazole aniline promotes dendritic spine formation through the RasGRF1-Ras dependent pathway.

Our recent study demonstrated that an amyloid-β binding molecule, BTA-EG4, increases dendritic spine number via Ras-mediated signaling. To potentially optimize the potency of the BTA compounds, we synthesized and evaluated an amyloid-β binding analog of BTA-EG4 with increased solubility in aqueous solution, BTA-EG6. We initially examined the effects of BTA-EG6 on dendritic spine formation and found that BTA-EG6-treated primary hippocampal neurons had significantly increased dendritic spine number compared to control treatment. In addition, BTA-EG6 significantly increased the surface level of AMPA receptors. Upon investigation into the molecular mechanism by which BTA-EG6 promotes dendritic spine formation, we found that BTA-EG6 may exert its effects on spinogenesis via RasGRF1-ERK signaling, with potential involvement of other spinogenesis-related proteins such as Cdc42 and CDK5. Taken together, our data suggest that BTA-EG6 boosts spine and synapse number, which may have a beneficial effect of enhancing neuronal and synaptic function in the normal healthy brain