12 research outputs found
Targeted rescue of cancer-associated IDH1 mutant activity using an engineered synthetic antibody
We have utilized a high-diversity phage display library to engineer antibody fragments (Fabs) that can modulate the activity of the enzyme isocitrate dehydrogenase 1 (IDH1). We show that a conformation-specific Fab can reactivate an IDH1 mutant associated with brain tumors. The results show that this strategy is a first step towards developing "activator drugs" for a large number of genetic disorders where mutations have disrupted protein function
Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling
Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how
each hormone binding event triggers the appropriate response.
Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.
Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity. Keywords: Prolactin signaling, Synthetic antibody, Phage display, Alloster
A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria
Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination
Molecular Modeling and Simulations of the Transmembrane Domain of Human Growth Hormone Receptor
Substance P Derivatives as Versatile Tools for Specific Delivery of Various Types of Biomolecular Cargo
The use of proteins or nucleic acids as therapeutic agents
has
been severely hampered by their intrinsic inability to cross the cell
membrane. Moreover, common techniques for driving the delivery of
macromolecules lack the ability to distinguish between healthy and
diseased tissue, precluding their clinical use. Recently, receptor-mediated
delivery (RMD) has emerged as a technology with the potential to circumvent
the obstacles associated with the delivery of drug targets by utilizing
the natural endocytosis of a ligand upon binding to its receptor.
Here, we describe the synthesis
of variants of substance P (SP), an eleven amino acid neuropeptide
ligand of the neurokinin type 1 receptor (NK1R), for the delivery
of various types of cargo. The variants of SP were synthesized with
an N-terminal maleimide moiety that allows conjugation to surface
thiols, resulting in a nonreducible thioether. Cargos lacking an available
thiol are conjugated to SP using commercially available cross-linkers.
In addition to the delivery of proteins, we expand the use of SP to
include nuclear delivery of DNA fragments that are actively expressed
in the target cells. We also show that SP can be used to deliver whole
bacteriophage particles as well as polystyrene beads up to 1 μm
in diameter. The results show the ability of SP to deliver cargo of
various sizes and chemical properties that retain their function within
the cell. Furthermore, the overexpression of the NK1R in many tumors
provides the potential for developing targeted delivery reagents that
are specific toward diseased tissue
Substance P Derivatives as Versatile Tools for Specific Delivery of Various Types of Biomolecular Cargo
The use of proteins or nucleic acids as therapeutic agents
has
been severely hampered by their intrinsic inability to cross the cell
membrane. Moreover, common techniques for driving the delivery of
macromolecules lack the ability to distinguish between healthy and
diseased tissue, precluding their clinical use. Recently, receptor-mediated
delivery (RMD) has emerged as a technology with the potential to circumvent
the obstacles associated with the delivery of drug targets by utilizing
the natural endocytosis of a ligand upon binding to its receptor.
Here, we describe the synthesis
of variants of substance P (SP), an eleven amino acid neuropeptide
ligand of the neurokinin type 1 receptor (NK1R), for the delivery
of various types of cargo. The variants of SP were synthesized with
an N-terminal maleimide moiety that allows conjugation to surface
thiols, resulting in a nonreducible thioether. Cargos lacking an available
thiol are conjugated to SP using commercially available cross-linkers.
In addition to the delivery of proteins, we expand the use of SP to
include nuclear delivery of DNA fragments that are actively expressed
in the target cells. We also show that SP can be used to deliver whole
bacteriophage particles as well as polystyrene beads up to 1 μm
in diameter. The results show the ability of SP to deliver cargo of
various sizes and chemical properties that retain their function within
the cell. Furthermore, the overexpression of the NK1R in many tumors
provides the potential for developing targeted delivery reagents that
are specific toward diseased tissue