Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins - BRD2, BRD3, BRD4 and BRDT ...bind acetylated chromatin
their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound - 9-ME-1 - shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of
and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and
target validation studies.
Molecular glues and bivalent inducers of protein degradation (also known as PROTACs) represent a fascinating new modality in pharmacotherapeutics: the potential to knockdown previously thought ...'undruggable' targets at sub-stoichiometric concentrations in ways not possible using conventional inhibitors. Mounting evidence suggests these chemical agents, in concert with their target proteins, can be modelled as three-body binding equilibria that can exhibit significant cooperativity as a result of specific ligand-induced molecular recognition. Despite this, many existing drug design and optimization regimens still fixate on binary target engagement, in part due to limited structural data on ternary complexes. Recent crystal structures of protein complexes mediated by degrader molecules, including the first PROTAC ternary complex, underscore the importance of protein-protein interactions and intramolecular contacts to the mode of action of this class of compounds. These discoveries have opened the door to a new paradigm for structure-guided drug design: borrowing surface area and molecular recognition from nature to elicit cellular signalling.
Knowledge of biomolecular interactions is of importance to our understanding of biological processes such as enzyme catalysis and inhibition. Biophysical techniques enable sensitive detection and ...accurate characterization of binding and are therefore powerful tools in enzymology and rational drug design. The applications of NMR spectroscopy and isothermal titration calorimetry to study enzyme-ligand interactions will be discussed. Recent work on ketopantoate reductase, which catalyses an important step on the biosynthetic pathway to vitamin B5, is used to illustrate the potential of this approach.
•We study the influence of host galaxy morphology on LC parameters of the JLA data.•The α parameter decreases from elliptical/lenticular to late-type spiral galaxies.•In an old stellar population SNe ...Ia are fainter after stretch and colour corrections.•The host galaxy morphology affects the residual dispersion on the Hubble diagram.•SNe Ia in late-type spiral galaxies are more homogeneous in comparison with others.
The observational cosmology with distant Type Ia supernovae (SNe) as standard candles claims that the Universe is in accelerated expansion, caused by a large fraction of dark energy. In this paper we investigate the SN Ia environment, studying the impact of the nature of their host galaxies on the Hubble diagram fitting. The supernovae (192 SNe) used in the analysis were extracted from Joint-Light-curves-Analysis (JLA) compilation of high-redshift and nearby supernovae which is the best one to date. The analysis is based on the empirical fact that SN Ia luminosities depend on their light curve shapes and colors. We confirm that the stretch parameter of Type Ia supernovae is correlated with the host galaxy type. The supernovae with lower stretch are hosted mainly in elliptical and lenticular galaxies. No significant correlation between SN Ia colour and host morphology was found.
We also examine how the luminosities of SNe Ia change depending on host galaxy morphology after stretch and colour corrections. Our results show that in old stellar populations and low dust environments, the supernovae are slightly fainter. SNe Ia in elliptical and lenticular galaxies have a higher α (slope in luminosity-stretch) and β (slope in luminosity-colour) parameter than in spirals. However, the observed shift is at the 1-σ uncertainty level and, therefore, can not be considered as significant.
We confirm that the supernova properties depend on their environment and that the incorporation of a host galaxy term into the Hubble diagram fit is expected to be crucial for future cosmological analyses.
Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins - BRD2, BRD3, BRD4 and BRDT ...bind acetylated chromatin
via
their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound - 9-ME-1 - shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of
in vitro
and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and
in vivo
target validation studies.
Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins.
Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins.
Allele-specific chemical genetics enables selective inhibition within families of ...highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins – BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin
via
their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure–activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound – 9-ME-1 – shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of
in vitro
and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and
in vivo
target validation studies.
Context. Type Ia supernovae (SNe Ia) are widely used to measure the expansion of the Universe. To perform such measurements the luminosity and cosmological redshift (z) of the SNe Ia have to be ...determined. The uncertainty on z includes an unknown peculiar velocity, which can be very large for SNe Ia in the virialized cores of massive clusters. Aims. We determine which SNe Ia exploded in galaxy clusters using 145 SNe Ia from the Nearby Supernova Factory. We then study how the correction for peculiar velocities of host galaxies inside the clusters improves the Hubble residuals. Methods. We found 11 candidates for membership in clusters. We applied the biweight technique to estimate the redshift of a cluster. Then, we used the galaxy cluster redshift instead of the host galaxy redshift to construct the Hubble diagram. Results. For SNe Ia inside galaxy clusters, the dispersion around the Hubble diagram when peculiar velocities are taken into account is smaller compared with a case without peculiar velocity correction, which has a wRMS = 0.130 +/- 0.038 mag instead of wRMS = 0.137 +/- 0.036 mag. The significance of this improvement is 3.58 sigma. If we remove the very nearby Virgo cluster member SN2006X (z < 0.01) from the analysis, the significance decreases to 1.34 sigma. The peculiar velocity correction is found to be highest for the SNe Ia hosted by blue spiral galaxies. Those SNe Ia have high local specific star formation rates and smaller stellar masses, which is seemingly counter to what might be expected given the heavy concentration of old, massive elliptical galaxies in clusters. Conclusions. As expected, the Hubble residuals of SNe Ia associated with massive galaxy clusters improve when the cluster redshift is taken as the cosmological redshift of the supernova. This fact has to be taken into account in future cosmological analyses in order to achieve higher accuracy for cosmological redshift measurements. We provide an approach to do so.
Context. Type Ia supernovae (SNe Ia) are widely used to measure the expansion of the Universe. To perform such measurements the luminosity and cosmological redshift (z) of the SNe Ia have to be ...determined. The uncertainty on z includes an unknown peculiar velocity, which can be very large for SNe Ia in the virialized cores of massive clusters. Aims. We determine which SNe Ia exploded in galaxy clusters using 145 SNe Ia from the Nearby Supernova Factory. We then study how the correction for peculiar velocities of host galaxies inside the clusters improves the Hubble residuals. Methods. We found 11 candidates for membership in clusters. We applied the biweight technique to estimate the redshift of a cluster. Then, we used the galaxy cluster redshift instead of the host galaxy redshift to construct the Hubble diagram. Results. For SNe Ia inside galaxy clusters, the dispersion around the Hubble diagram when peculiar velocities are taken into account is smaller compared with a case without peculiar velocity correction, which has a wRMS = 0.130 ± 0.038 mag instead of wRMS = 0.137 ± 0.036 mag. The significance of this improvement is 3.58σ. If we remove the very nearby Virgo cluster member SN2006X (z < 0.01) from the analysis, the significance decreases to 1.34σ. The peculiar velocity correction is found to be highest for the SNe Ia hosted by blue spiral galaxies. Those SNe Ia have high local specific star formation rates and smaller stellar masses, which is seemingly counter to what might be expected given the heavy concentration of old, massive elliptical galaxies in clusters. Conclusions. As expected, the Hubble residuals of SNe Ia associated with massive galaxy clusters improve when the cluster redshift is taken as the cosmological redshift of the supernova. This fact has to be taken into account in future cosmological analyses in order to achieve higher accuracy for cosmological redshift measurements. We provide an approach to do so.
Proteolysis Targeting Chimaeras (PROTACs) offer a powerful strategy to degrade cancer-associated proteins. PROTACs recruit an E3 ubiquitin ligase, such as von Hippel-Lindau (VHL), to a protein of ...interest (POI) to form a ternary complex. Measuring the formation of this ternary species is important for understanding PROTAC mode of action. Proximity AlphaLISA assays can be used for this purpose but have, to date, focused on using purified recombinant protein in vitro. Here, we report the development and optimisation of an AlphaLISA assay for monitoring ternary complex formation using full-length POI within cell lysates.
AlphaLISA assays were used to profile PROTAC-induced complex formation of FLAG-tagged full-length POI in cell lysate to purified biotinylated VCB. Signal was measured from close proximity of FLAG-tagged acceptor beads to streptavidin donor beads and benchmarked against a well-characterised PROTAC, MZ1, purified His-tagged Brd4 bromodomain 2 (BD2) and biotinylated VCB. A FLAG-tagged full-length POI containing an insert of Brd4 BD2 (POI-fusion) was utilised, such that MZ1 could be employed as a tool compound for assay development.
Signal intensity was dependent on total lysate protein and VCB concentration, allowing for optimisation of component concentrations for maximal signal. A mild, detergent-free hypotonic cell lysis buffer gave enhanced signal over harsher buffers containing detergents. Additionally, the cell lysate cytosolic fraction provided superior signal compared to nuclear fractions and whole cell lysates, further highlighting the reliance of transient ternary complex formation on assay conditions. By utilising these optimal assay conditions determined with the POI-fusion, it is now possible to detect ternary complex formation between POI-targeting PROTACs and full-length POI.
To our knowledge, this optimised cell lysate AlphaLISA assay provides the first report and proof of concept for the interrogation of PROTAC-induced complex formation of full-length proteins with E3 ligases, which will be a valuable tool to the burgeoning PROTAC field. Consideration of assay parameters that permit optimal conditions for ternary complex formation in a POI-dependent manner allows the application of the AlphaLISA method as a screening platform. This application could profile PROTAC efficiency for a wide range of full-length, cell-derived proteins that are more relevant to the endogenous context encountered by the PROTAC intracellularly.
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