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A molecular tweezer antagonizes seminal amyloids and HIV infection.

Lump E, Castellano LM, Meier C, Seeliger J, Erwin N, Sperlich B, Stürzel CM, Usmani S, Hammond RM, von Einem J, Gerold G, Kreppel F, Bravo-Rodriguez K, Pietschmann T, Holmes VM, Palesch D, Zirafi O, Weissman D, Sowislok A, Wettig B, Heid C, Kirchhoff F, Weil T, Klärner FG, Schrader T, Bitan G, Sanchez-Garcia E, Winter R, Shorter J, Münch J - Elife (2015)

Bottom Line: In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils.We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism.CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

ABSTRACT
Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

No MeSH data available.


Related in: MedlinePlus

Stejskal and Tanner Plot.Signal intensity in diffusion NMR experiments obeys the following law: ln(I/I0) = −γ2δ2G2(∆ − δ/3) D, where I = intensity at a given G, I0 = Intensity at G = 0, γ = Gyromagnetic ratio (for 1H: γ = 2.675 × 108T−1s−1), δ = Length of diffusion gradient, ∆ = Diffusion shift, G = Gradient field intensity, and D = Diffusion coefficient. Signal intensities are used to generate a Stejskal Tanner-Plot lg(I/I0) vs G2. Its slope yields the diffusion coefficient. Diffusion coefficient for CLR01 (6.4 mM in PBS): 2.433 × 10−10 m2s−1. By means of the Stokes Einstein equation one calculates the hydrodynamic radius: r(s) = (k*T)/(6*pi*η*D). The hydrodynamic radius for CLR01 (6.4 mM) was determined at 1.0075 × 10−9 m ∼10 Å in PBS buffer; for CLR01 (0.2 mM) the diffusion coefficient dropped to 2.702 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.072 × 10−9 m ∼9 Å. In HEPES buffer, the diffusion coefficient for CLR01 (2.0 mM) was determined at 2.449 × 10−10 m2s−1, corresponding to hydrodynamic radius r(s) of 1.001 × 10−9 m ∼10 Å; for CLR01 (0.5 mM) the diffusion coefficient dropped to 2.579 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.504 × 10−9 m ∼9 Å. In summary, almost identical r(s) values were found for PBS and HEPES buffer. Thus, at various concentrations even above those used in experiments, the tweezers only produced a hydrodynamic radius slightly above the monomeric species.DOI:http://dx.doi.org/10.7554/eLife.05397.026
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fig7s3: Stejskal and Tanner Plot.Signal intensity in diffusion NMR experiments obeys the following law: ln(I/I0) = −γ2δ2G2(∆ − δ/3) D, where I = intensity at a given G, I0 = Intensity at G = 0, γ = Gyromagnetic ratio (for 1H: γ = 2.675 × 108T−1s−1), δ = Length of diffusion gradient, ∆ = Diffusion shift, G = Gradient field intensity, and D = Diffusion coefficient. Signal intensities are used to generate a Stejskal Tanner-Plot lg(I/I0) vs G2. Its slope yields the diffusion coefficient. Diffusion coefficient for CLR01 (6.4 mM in PBS): 2.433 × 10−10 m2s−1. By means of the Stokes Einstein equation one calculates the hydrodynamic radius: r(s) = (k*T)/(6*pi*η*D). The hydrodynamic radius for CLR01 (6.4 mM) was determined at 1.0075 × 10−9 m ∼10 Å in PBS buffer; for CLR01 (0.2 mM) the diffusion coefficient dropped to 2.702 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.072 × 10−9 m ∼9 Å. In HEPES buffer, the diffusion coefficient for CLR01 (2.0 mM) was determined at 2.449 × 10−10 m2s−1, corresponding to hydrodynamic radius r(s) of 1.001 × 10−9 m ∼10 Å; for CLR01 (0.5 mM) the diffusion coefficient dropped to 2.579 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.504 × 10−9 m ∼9 Å. In summary, almost identical r(s) values were found for PBS and HEPES buffer. Thus, at various concentrations even above those used in experiments, the tweezers only produced a hydrodynamic radius slightly above the monomeric species.DOI:http://dx.doi.org/10.7554/eLife.05397.026

Mentions: Diffusion NMR (DOSY) experiments in both buffers revealed CLR01 hydrodynamic radii of ∼0.9–1.0 nm, slightly above the monomeric species (Figure 7C,D, Figure 7—figure supplement 3). Microcalorimetric dilution titrations revealed only minute endothermic heat changes, which argue strongly against an extensive aggregation process (data not shown). Pyrene fluorescence revealed that no critical micelle concentration (cmc) could be determined in a wide concentration range (0–0.5 mM CLR01) encompassing fibril assembly and remodeling conditions (Figure 7E). Dynamic light scattering (DLS) experiments showed CLR01 particles with hydrodynamic radius RH = 5–8 nm in PBS at concentration between 200 and 1000 μM, plus a very minor fraction of particles with a RH = 20–40 nm (Figure 7F; data not shown). We did not observe CLR01 particles with a RH of ∼95–400 nm, which is the size range typically associated with colloidal small-molecule aggregates (McGovern et al., 2002). No CLR01 particles could be detected at 10 or 50 μM in PBS (data not shown). In HEPES buffer, no particles were detected at any of the concentration tested (between 10 and 1000 μM) (Figure 7F; data not shown). The ∼0.9–1 nm hydrodynamic radius of CLR01 revealed by DOSY (Figure 7C,D) is below the detection limit of our DLS instrument, and hence, we cannot resolve the CLR01 monomer (Figure 7F). Importantly, DLS overemphasizes large particles because the scattered-light intensity is proportional to the square of the particle mass. The scattering intensity in the solutions of CLR01 (1 mM) in PBS was ∼3% that of similar samples containing Mg3(PO4) colloids (10 mM Mg3(PO4)) or SDS micelles (2% SDS (wt/vol)), suggesting that the observed species represented a small fraction of the CLR01 molecules (data not shown). Thus, under all assay concentrations the vast majority of the tweezer molecules are monomeric. Minute amounts of dimers or higher order species may be present at high concentrations of CLR01 in some cases in PBS (fibril assembly buffer) but are absent from the HEPES buffer (fibril remodeling buffer). Thus, it is highly unlikely that colloidal CLR01 aggregates contribute to the observed activity of the tweezer.


A molecular tweezer antagonizes seminal amyloids and HIV infection.

Lump E, Castellano LM, Meier C, Seeliger J, Erwin N, Sperlich B, Stürzel CM, Usmani S, Hammond RM, von Einem J, Gerold G, Kreppel F, Bravo-Rodriguez K, Pietschmann T, Holmes VM, Palesch D, Zirafi O, Weissman D, Sowislok A, Wettig B, Heid C, Kirchhoff F, Weil T, Klärner FG, Schrader T, Bitan G, Sanchez-Garcia E, Winter R, Shorter J, Münch J - Elife (2015)

Stejskal and Tanner Plot.Signal intensity in diffusion NMR experiments obeys the following law: ln(I/I0) = −γ2δ2G2(∆ − δ/3) D, where I = intensity at a given G, I0 = Intensity at G = 0, γ = Gyromagnetic ratio (for 1H: γ = 2.675 × 108T−1s−1), δ = Length of diffusion gradient, ∆ = Diffusion shift, G = Gradient field intensity, and D = Diffusion coefficient. Signal intensities are used to generate a Stejskal Tanner-Plot lg(I/I0) vs G2. Its slope yields the diffusion coefficient. Diffusion coefficient for CLR01 (6.4 mM in PBS): 2.433 × 10−10 m2s−1. By means of the Stokes Einstein equation one calculates the hydrodynamic radius: r(s) = (k*T)/(6*pi*η*D). The hydrodynamic radius for CLR01 (6.4 mM) was determined at 1.0075 × 10−9 m ∼10 Å in PBS buffer; for CLR01 (0.2 mM) the diffusion coefficient dropped to 2.702 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.072 × 10−9 m ∼9 Å. In HEPES buffer, the diffusion coefficient for CLR01 (2.0 mM) was determined at 2.449 × 10−10 m2s−1, corresponding to hydrodynamic radius r(s) of 1.001 × 10−9 m ∼10 Å; for CLR01 (0.5 mM) the diffusion coefficient dropped to 2.579 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.504 × 10−9 m ∼9 Å. In summary, almost identical r(s) values were found for PBS and HEPES buffer. Thus, at various concentrations even above those used in experiments, the tweezers only produced a hydrodynamic radius slightly above the monomeric species.DOI:http://dx.doi.org/10.7554/eLife.05397.026
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Related In: Results  -  Collection

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fig7s3: Stejskal and Tanner Plot.Signal intensity in diffusion NMR experiments obeys the following law: ln(I/I0) = −γ2δ2G2(∆ − δ/3) D, where I = intensity at a given G, I0 = Intensity at G = 0, γ = Gyromagnetic ratio (for 1H: γ = 2.675 × 108T−1s−1), δ = Length of diffusion gradient, ∆ = Diffusion shift, G = Gradient field intensity, and D = Diffusion coefficient. Signal intensities are used to generate a Stejskal Tanner-Plot lg(I/I0) vs G2. Its slope yields the diffusion coefficient. Diffusion coefficient for CLR01 (6.4 mM in PBS): 2.433 × 10−10 m2s−1. By means of the Stokes Einstein equation one calculates the hydrodynamic radius: r(s) = (k*T)/(6*pi*η*D). The hydrodynamic radius for CLR01 (6.4 mM) was determined at 1.0075 × 10−9 m ∼10 Å in PBS buffer; for CLR01 (0.2 mM) the diffusion coefficient dropped to 2.702 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.072 × 10−9 m ∼9 Å. In HEPES buffer, the diffusion coefficient for CLR01 (2.0 mM) was determined at 2.449 × 10−10 m2s−1, corresponding to hydrodynamic radius r(s) of 1.001 × 10−9 m ∼10 Å; for CLR01 (0.5 mM) the diffusion coefficient dropped to 2.579 × 10−10 m2s−1, corresponding to a hydrodynamic radius of 9.504 × 10−9 m ∼9 Å. In summary, almost identical r(s) values were found for PBS and HEPES buffer. Thus, at various concentrations even above those used in experiments, the tweezers only produced a hydrodynamic radius slightly above the monomeric species.DOI:http://dx.doi.org/10.7554/eLife.05397.026
Mentions: Diffusion NMR (DOSY) experiments in both buffers revealed CLR01 hydrodynamic radii of ∼0.9–1.0 nm, slightly above the monomeric species (Figure 7C,D, Figure 7—figure supplement 3). Microcalorimetric dilution titrations revealed only minute endothermic heat changes, which argue strongly against an extensive aggregation process (data not shown). Pyrene fluorescence revealed that no critical micelle concentration (cmc) could be determined in a wide concentration range (0–0.5 mM CLR01) encompassing fibril assembly and remodeling conditions (Figure 7E). Dynamic light scattering (DLS) experiments showed CLR01 particles with hydrodynamic radius RH = 5–8 nm in PBS at concentration between 200 and 1000 μM, plus a very minor fraction of particles with a RH = 20–40 nm (Figure 7F; data not shown). We did not observe CLR01 particles with a RH of ∼95–400 nm, which is the size range typically associated with colloidal small-molecule aggregates (McGovern et al., 2002). No CLR01 particles could be detected at 10 or 50 μM in PBS (data not shown). In HEPES buffer, no particles were detected at any of the concentration tested (between 10 and 1000 μM) (Figure 7F; data not shown). The ∼0.9–1 nm hydrodynamic radius of CLR01 revealed by DOSY (Figure 7C,D) is below the detection limit of our DLS instrument, and hence, we cannot resolve the CLR01 monomer (Figure 7F). Importantly, DLS overemphasizes large particles because the scattered-light intensity is proportional to the square of the particle mass. The scattering intensity in the solutions of CLR01 (1 mM) in PBS was ∼3% that of similar samples containing Mg3(PO4) colloids (10 mM Mg3(PO4)) or SDS micelles (2% SDS (wt/vol)), suggesting that the observed species represented a small fraction of the CLR01 molecules (data not shown). Thus, under all assay concentrations the vast majority of the tweezer molecules are monomeric. Minute amounts of dimers or higher order species may be present at high concentrations of CLR01 in some cases in PBS (fibril assembly buffer) but are absent from the HEPES buffer (fibril remodeling buffer). Thus, it is highly unlikely that colloidal CLR01 aggregates contribute to the observed activity of the tweezer.

Bottom Line: In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils.We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism.CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

ABSTRACT
Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

No MeSH data available.


Related in: MedlinePlus