6 research outputs found
Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure
The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.European Research Council (ERC) AdG 745225Swedish Research Council 541-2013-8351Publishe
Structure and characterization of a high affinity C5a monoclonal antibody that blocks binding to C5aR1 and C5aR2 receptors
Exploration of the HIF-1α/p300 interface using peptide and Adhiron phage display technologies.
The HIF-1α/p300 protein-protein interaction plays a key role in tumor metabolism and thus represents a high value target for anticancer drug-development. Although several studies have identified inhibitor candidates using rationale design, more detailed understanding of the interaction and binding interface is necessary to inform development of superior inhibitors. In this work, we report a detailed biophysical analysis of the native interaction with both peptide and Adhiron phage display experiments to identify novel binding motifs and binding regions of the surface of p300 to inform future inhibitor design
Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl‑1 Inhibitors
Mcl-1 is a pro-apoptotic
BH3 protein family member similar to Bcl-2
and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors
and allows cancer cells to evade apoptosis. Here we report the discovery
and optimization of a series of non-natural peptide Mcl-1 inhibitors.
Screening of DNA-encoded libraries resulted in hit compound <b>1</b>, a 1.5 ÎĽM Mcl-1 inhibitor. A subsequent crystal structure
demonstrated that compound <b>1</b> bound to Mcl-1 in a β-turn
conformation, such that the two ends of the peptide were close together.
This proximity allowed for the linking of the two ends of the peptide
to form a macrocycle. Macrocyclization resulted in an approximately
10-fold improvement in binding potency. Further exploration of a key
hydrophobic interaction with Mcl-1 protein and also with the moiety
that engages Arg256 led to additional potency improvements. The use
of protein–ligand crystal structures and binding kinetics contributed
to the design and understanding of the potency gains. Optimized compound <b>26</b> is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at
only 5 ÎĽM and Bcl-xL at >99 ÎĽM, and induces cleaved
caspase-3
in MV4–11 cells with an IC<sub>50</sub> of 3 μM after
6 h
Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl‑1 Inhibitors
Mcl-1 is a pro-apoptotic
BH3 protein family member similar to Bcl-2
and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors
and allows cancer cells to evade apoptosis. Here we report the discovery
and optimization of a series of non-natural peptide Mcl-1 inhibitors.
Screening of DNA-encoded libraries resulted in hit compound <b>1</b>, a 1.5 ÎĽM Mcl-1 inhibitor. A subsequent crystal structure
demonstrated that compound <b>1</b> bound to Mcl-1 in a β-turn
conformation, such that the two ends of the peptide were close together.
This proximity allowed for the linking of the two ends of the peptide
to form a macrocycle. Macrocyclization resulted in an approximately
10-fold improvement in binding potency. Further exploration of a key
hydrophobic interaction with Mcl-1 protein and also with the moiety
that engages Arg256 led to additional potency improvements. The use
of protein–ligand crystal structures and binding kinetics contributed
to the design and understanding of the potency gains. Optimized compound <b>26</b> is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at
only 5 ÎĽM and Bcl-xL at >99 ÎĽM, and induces cleaved
caspase-3
in MV4–11 cells with an IC<sub>50</sub> of 3 μM after
6 h