9 research outputs found
Multivalent biological interactions for the detection and inhibition of HIV-1 protease
Several diseases including cancer and pathogen infection are mediated by protease activity. In HIV infection, the viral protease plays a central role in the virus lifecycle, which has made it a clear therapeutic target. The dominant approach for the treatment of HIV is heavily dependent on inhibitors of this enzyme, but no new drugs have reached the market since 2006. There is thus a need for new design principles for the development of anti-retroviral therapies. Traditional methods of HIV detection are also limited in their use at point-of-care in resource-limited settings due to their reliance on highly trained laboratory personnel, cold-chain transport and expensive reagents.
This thesis examines the role of peptide-protein interactions for the inhibition and detection of HIV-1 protease. Phage display is used to isolate heptameric peptide sequences which interact specifically with the enzyme. These peptides are then utilised as sensors for the detection of the enzyme through Forster Resonance Energy Transfer (FRET). The inhibitory properties of the
peptides, both in isolation and through multivalent conjugates are also investigated.
Finally, insights into the nature of these peptide-protein interactions are explored through molecular docking and all-atom classical molecular dynamics simulations. The expression of recombinant HIV-1 protease in E. coli is
also discussed. The peptide based systems described here are expected to be more stable to environmental effects than protein based therapies and it is hoped that this work will provide new pathways for the design of peptide-based
therapeutics and diagnostics for protease related diseases which do not rely on traditional methods.Open Acces
Highly Controlled Open Vessel RAFT Polymerizations by Enzyme Degassing
Intolerance to oxygen is a key limitation
in many reactions and
particularly in controlled radical polymerizations. Here we introduce
the use of enzymes such as glucose oxidase (GOx) to deoxygenate reversible
additionâfragmentation chain transfer polymerizations (Enz-RAFT),
facilitating the preparation of highly controlled polymers in vessels
open to ambient oxygen. Because the removal of oxygen is so efficient,
very low concentrations of GOx and initiator can be used, enabling
excellent control which is demonstrated by pseudoliving polymerization
kinetics and the preparation of multiblock copolymers with narrow
molecular weight distributions (<i>M</i><sub>w</sub>/<i>M</i><sub>n</sub> < 1.15). GOx retains sufficient activity
to facilitate polymerization not only in aqueous solutions but also
in a range of water/organic solvent mixtures, and we demonstrate the
use of this technique to perform open vessel Enz-RAFT polymerizations
in various methanol and dioxane/water mixtures
Alcune osservazioni sul gerundio in italiano e tedesco
HIV-1 protease is a key enzyme in the life cycle of HIV/AIDS, as it is responsible for the formation of the mature virus particle. We demonstrate here that phage-display peptides raised against this enzyme can be used as peptide sensors for the detection of HIV-1 protease in a simple, one-pot assay. The presence of the enzyme is detected through an energy transfer between two peptide sensors when simultaneously complexed with the target protein. The multivalent nature of this assay increases the specificity of the detection by requiring all molecules to be interacting in order for there to be a FRET signal. We also perform molecular dynamics simulations to explore the interaction between the protease and the peptides in order to guide the design of these peptide sensors and to understand the mechanisms which cause these simultaneous binding events. This approach aims to facilitate the development of new assays for enzymes that are not dependent on the cleavage of a substrate and do not require multiple washing steps
Designing Fluorescent Peptide Sensors with Dual Specificity for the Detection of HIV-1 Protease
Fate of Liposomes in the Presence of Phospholipase C and D:From 2 Atomic to Supramolecular Lipid Arrangement
Understanding the
origins of lipid membrane bilayer rearrangement
in response to external stimuli is an essential component of cell
biology and the bottom-up design of liposomes for biomedical applications.
The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorusâoxygen
bonds of phosphate esters in phosphatidylcholine (PC) lipids. The
atomic position of this hydrolysis reaction has huge implications
for the stability of PC-containing self-assembled structures, such
as the cell wall and lipid-based vesicle drug delivery vectors. While
PLC converts PC to diacylglycerol (DAG), the interaction of PC with
PLD produces phosphatidic acid (PA). Here we present a combination
of small-angle scattering data and all-atom molecular dynamics simulations,
providing insights into the effects of atomic-scale reorganization
on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated
incorporation of DAG or PA. We observed that PC liposomes completely
disintegrate in the presence of PLC, as conversion of PC to DAG progresses.
At lower concentrations, DAG molecules within fluid PC bilayers form
hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules
and burrow into the hydrophobic region. This leads initially to membrane
thinning followed by a swelling of the lamellar phase with increased
DAG. At higher DAG concentrations, localized membrane tension causes
a change in lipid phase from lamellar to the hexagonal and micellar
cubic phases. Molecular dynamics simulations show that this destabilization
is also caused in part by the decreased ability of DAG-containing
PC membranes to coordinate sodium ions. Conversely, PLD-treated PC
liposomes remain stable up to extremely high conversions to PA. Here,
the negatively charged PA headgroup attracts significant amounts of
sodium ions from the bulk solution to the membrane surface, leading
to a swelling of the coordinated water layer. These findings are a
vital step toward a fundamental understanding of the degradation behavior
of PC lipid membranes in the presence of these clinically relevant
enzymes, and toward the rational design of diagnostic and drug delivery
technologies for phospholipase-dysregulation-based diseases
Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement
Understanding the
origins of lipid membrane bilayer rearrangement
in response to external stimuli is an essential component of cell
biology and the bottom-up design of liposomes for biomedical applications.
The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorusâoxygen
bonds of phosphate esters in phosphatidylcholine (PC) lipids. The
atomic position of this hydrolysis reaction has huge implications
for the stability of PC-containing self-assembled structures, such
as the cell wall and lipid-based vesicle drug delivery vectors. While
PLC converts PC to diacylglycerol (DAG), the interaction of PC with
PLD produces phosphatidic acid (PA). Here we present a combination
of small-angle scattering data and all-atom molecular dynamics simulations,
providing insights into the effects of atomic-scale reorganization
on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated
incorporation of DAG or PA. We observed that PC liposomes completely
disintegrate in the presence of PLC, as conversion of PC to DAG progresses.
At lower concentrations, DAG molecules within fluid PC bilayers form
hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules
and burrow into the hydrophobic region. This leads initially to membrane
thinning followed by a swelling of the lamellar phase with increased
DAG. At higher DAG concentrations, localized membrane tension causes
a change in lipid phase from lamellar to the hexagonal and micellar
cubic phases. Molecular dynamics simulations show that this destabilization
is also caused in part by the decreased ability of DAG-containing
PC membranes to coordinate sodium ions. Conversely, PLD-treated PC
liposomes remain stable up to extremely high conversions to PA. Here,
the negatively charged PA headgroup attracts significant amounts of
sodium ions from the bulk solution to the membrane surface, leading
to a swelling of the coordinated water layer. These findings are a
vital step toward a fundamental understanding of the degradation behavior
of PC lipid membranes in the presence of these clinically relevant
enzymes, and toward the rational design of diagnostic and drug delivery
technologies for phospholipase-dysregulation-based diseases
Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement
Understanding the
origins of lipid membrane bilayer rearrangement
in response to external stimuli is an essential component of cell
biology and the bottom-up design of liposomes for biomedical applications.
The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorusâoxygen
bonds of phosphate esters in phosphatidylcholine (PC) lipids. The
atomic position of this hydrolysis reaction has huge implications
for the stability of PC-containing self-assembled structures, such
as the cell wall and lipid-based vesicle drug delivery vectors. While
PLC converts PC to diacylglycerol (DAG), the interaction of PC with
PLD produces phosphatidic acid (PA). Here we present a combination
of small-angle scattering data and all-atom molecular dynamics simulations,
providing insights into the effects of atomic-scale reorganization
on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated
incorporation of DAG or PA. We observed that PC liposomes completely
disintegrate in the presence of PLC, as conversion of PC to DAG progresses.
At lower concentrations, DAG molecules within fluid PC bilayers form
hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules
and burrow into the hydrophobic region. This leads initially to membrane
thinning followed by a swelling of the lamellar phase with increased
DAG. At higher DAG concentrations, localized membrane tension causes
a change in lipid phase from lamellar to the hexagonal and micellar
cubic phases. Molecular dynamics simulations show that this destabilization
is also caused in part by the decreased ability of DAG-containing
PC membranes to coordinate sodium ions. Conversely, PLD-treated PC
liposomes remain stable up to extremely high conversions to PA. Here,
the negatively charged PA headgroup attracts significant amounts of
sodium ions from the bulk solution to the membrane surface, leading
to a swelling of the coordinated water layer. These findings are a
vital step toward a fundamental understanding of the degradation behavior
of PC lipid membranes in the presence of these clinically relevant
enzymes, and toward the rational design of diagnostic and drug delivery
technologies for phospholipase-dysregulation-based diseases