5 research outputs found
Generation of Thiocillin Ring Size Variants by Prepeptide Gene Replacement and in Vivo Processing by <i>Bacillus cereus</i>
The thiocillins from <i>Bacillus cereus</i> ATCC 14579
are natural products from the broader class of thiazolyl peptides.
Their biosynthesis proceeds via extensive post-translational modification
of a ribosomally encoded precursor peptide. This post-translational
tailoring involves a key step formal cycloaddition between two distal
serine residues. In the wild-type structure, this cycloaddition forms
a major macrocycle circumscribed by 26-atoms (shortest path). Results
presented herein demonstrate the promiscuity of this last step by
means of a set of “competition” experiments. Cyclization
proceeds in many cases to provide altered ring sizes, giving access
to several variant rings sizes that have not previously been observed
in nature
Ribosomal Route to Small-Molecule Diversity
The cyanobactin ribosomal peptide (RP) natural product
pathway
was manipulated to incorporate multiple tandem mutations and non-proteinogenic
amino acids, using eight heterologous components simultaneously expressed
in Escherichia coli. These studies
reveal the potential of RPs for the rational synthesis of complex,
new small molecules over multiple-step biosynthetic pathways using
simple genetic engineering
Structure–Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product
Macrocycles
are appealing drug candidates due to their high affinity,
specificity, and favorable pharmacological properties. In this study,
we explored the effects of chemical modifications to a natural product
macrocycle upon its activity, 3D geometry, and conformational entropy.
We chose thiocillin as a model system, a thiopeptide in the ribosomally
encoded family of natural products that exhibits potent antimicrobial
effects against Gram-positive bacteria. Since thiocillin is derived
from a genetically encoded peptide scaffold, site-directed mutagenesis
allows for rapid generation of analogues. To understand thiocillin’s
structure–activity relationship, we generated a site-saturation
mutagenesis library covering each position along thiocillin’s
macrocyclic ring. We report the identification of eight unique compounds
more potent than wild-type thiocillin, the best having an 8-fold improvement
in potency. Computational modeling of thiocillin’s macrocyclic
structure revealed a striking requirement for a low-entropy macrocycle
for activity. The populated ensembles of the active mutants showed
a rigid structure with few adoptable conformations while inactive
mutants showed a more flexible macrocycle which is unfavorable for
binding. This finding highlights the importance of macrocyclization
in combination with rigidifying post-translational modifications to
achieve high-potency binding
Redirection of Genetically Engineered CAR‑T Cells Using Bifunctional Small Molecules
Chimeric antigen receptor (CAR)-engineered
T cells (CAR-Ts) provide
a potent antitumor response and have become a promising treatment
option for cancer. However, despite their efficacy, CAR-T cells are
associated with significant safety challenges related to the inability
to control their activation and expansion and terminate their response.
Herein, we demonstrate that a bifunctional small molecule “switch”
consisting of folate conjugated to fluorescein isothiocyanate (folate-FITC)
can redirect and regulate FITC-specific CAR-T cell activity toward
folate receptor (FR)-overexpressing tumor cells. This system was shown
to be highly cytotoxic to FR-positive cells with no activity against
FR-negative cells, demonstrating the specificity of redirection by
folate-FITC. Anti-FITC-CAR-T cell activation and proliferation was
strictly dependent on the presence of both folate-FITC and FR-positive
cells and was dose titratable with folate-FITC switch. This novel
treatment paradigm may ultimately lead to increased safety for CAR-T
cell immunotherapy
An Immunosuppressive Antibody–Drug Conjugate
We have developed a novel antibody–drug
conjugate (ADC)
that can selectively deliver the Lck inhibitor dasatinib to human
T lymphocytes. This ADC is based on a humanized antibody that selectively
binds with high affinity to CXCR4, an antigen that is selectively
expressed on hematopoietic cells. The resulting dasatinib–antibody
conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation
and cytokine expression with low nM EC<sub>50</sub> and has minimal
effects on cell viability. This ADC may lead to a new class of selective
immunosuppressive drugs with improved safety and extend the ADC strategy
to the targeted delivery of kinase inhibitors for indications beyond
oncology