9 research outputs found
Linkage Isomerization Equilibria in (Chloranilato)palladium(II) Complexes with Amine Ligands. Crystal Structure of (Chloranilato)bis(acetonitrile)palladium(II)
Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies
As the antibody-drug conjugate (ADC)
field grows increasingly important
for cancer treatment, it is vital for researchers to establish a firm
understanding of how ADCs function at the molecular level. To gain
insight into ADC uptake, trafficking, and catabolismprocesses
that are critical to ADC efficacy and toxicityimaging studies
have been performed with fluorophore-labeled conjugates. However,
such labels may alter the properties and behavior of the ADC under
investigation. As an alternative approach, we present here the development
of a “clickable” ADC bearing an azide-functionalized
linker-payload (LP) poised for “click” reaction with
alkyne fluorophores; the azide group represents a significantly smaller
structural perturbation to the LP than most fluorophores. Notably,
the clickable ADC shows excellent potency in target-expressing cells,
whereas the fluorophore-labeled product ADC suffers from a significant
loss of activity, underscoring the impact of the label itself on the
payload. Live-cell confocal microscopy reveals robust uptake of the
clickable ADC, which reacts selectively in situ with a derivatized
fluorescent label. Time-course trafficking studies show greater and
more rapid net internalization of the ADCs than the parent antibody.
More generally, the application of chemical biology tools to the study
of ADCs should improve our understanding of how ADCs are processed
in biological systems
Deconstruction of Activity-Dependent Covalent Modification of Heme in Human Neutrophil Myeloperoxidase by Multistage Mass Spectrometry (MS<sup>4</sup>)
Myeloperoxidase (MPO) is known to be inactivated and
covalently
modified by treatment with hydrogen peroxide and agents similar to
3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1<i>H</i>-purin-6(9<i>H</i>)-one (<b>1</b>), a 254.08 Da derivative of 2-thioxanthine.
Peptide mapping by liquid chromatography and mass spectrometry detected
modification by <b>1</b> in a labile peptide–heme–peptide
fragment of the enzyme, accompanied by a mass increase of 252.08 Da.
The loss of two hydrogen atoms was consistent with mechanism-based
oxidative coupling. Multistage mass spectrometry (MS<sup>4</sup>)
of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated
that <b>1</b> was coupled directly to heme. Use of a 10 amu
window delivered the full isotopic envelope of each precursor ion
to collision-induced dissociation, preserving definitive isotopic
profiles for iron-containing fragments through successive steps of
multistage mass spectrometry. Iron isotope signatures and accurate
mass measurements supported the structural assignments. Crystallographic
analysis confirmed linkage between the methyl substituent of the heme
pyrrole D ring and the sulfur atom of <b>1</b>. The final orientation
of <b>1</b> perpendicular to the plane of the heme ring suggested
a mechanism consisting of two consecutive one-electron oxidations
of <b>1</b> by MPO. Multistage mass spectrometry using stage-specific
collision energies permits stepwise deconstruction of modifications
of heme enzymes containing covalent links between the heme group and
the polypeptide chain
Determination of Antibody–Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation
Antibody-drug conjugates
(ADC) are currently an active area of
research, focused primarily on oncology therapeutics, but also to
a limited extent on other areas such as infectious disease. The success
of this type of targeted drug delivery is dependent upon many factors,
one of which is the performance of the linker in releasing an active
drug moiety under the appropriate conditions. As a tool in the development
of linker/payload chemistry, we have developed an in vitro method
for the identification of payload species released from ADCs in the
presence of lysosomal enzymes. This method utilizes commercially available
human liver S9 fraction as the source of these enzymes, and this has
certain advantages over lysosomal fractions or purified enzymes. This
article describes the characterization and performance of this assay
with multiple ADCs composed of known and novel linkers and payloads.
Additionally, we report the observation of incomplete degradation
of mAb protein chains by lysosomal enzymes in vitro, believed to be
the first report of this phenomenon involving an ADC therapeutic
Optimization of Tubulysin Antibody–Drug Conjugates: A Case Study in Addressing ADC Metabolism
As part of our efforts to develop
new classes of tubulin inhibitor payloads for antibody–drug
conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated
excellent in vitro activity but suffered from rapid metabolism of
a critical acetate ester. A two-pronged strategy was employed to address
this metabolism. First, the hydrolytically labile ester was replaced
by a carbamate functional group resulting in a more stable ADC that
retained potency in cellular assays. Second, site-specific conjugation
was employed in order to design ADCs with reduced metabolic liabilities.
Using the later approach, we were able to identify a conjugate at
the 334C position of the heavy chain that resulted in an ADC with
considerably reduced metabolism and improved efficacy. The examples
discussed herein provide one of the clearest demonstrations to-date
that site of conjugation can play a critical role in addressing metabolic
and PK liabilities of an ADC. Moreover, a clear correlation was identified
between the hydrophobicity of an ADC and its susceptibility to metabolic
enzymes. Importantly, this study demonstrates that traditional medicinal
chemistry strategies can be effectively applied to ADC programs