Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-, and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.
We have recently developed an enzyme-directed immunostimulant (EDI) prodrug motif, which is metabolized to active immunostimulant by cancer cells and, following drug efflux, activates nearby immune cells, resulting in immunogenicity. In this study, we synthesized several EDI prodrugs featuring an imidazoquinoline immunostimulant resiquimod (a Toll-like receptor 7/8 agonist) covalently modified with glycosidase enzyme-directing groups selected from substrates of β-glucuronidase, α-mannosidase, or β-galactosidase. We compared the glycosidase-dependent immunogenicity elicited by each EDI in RAW-Blue macrophages following conversion to active immunostimulant by complementary glycosidase. At a cellular level, we examined EDI metabolism across three cancer cell lines (B16 melanoma, TC2 prostate, and 4T1 breast cancer). Comparing the relative immunogenicity elicited by each EDI/cancer cell combination, we found that B16 cells produced the highest EDI prodrug immunogenicity, achieving >95% of that elicited by unmodified resiquimod, followed by TC2 and 4T1 cells (40% and 30%, respectively). Immunogenicity elicited was comparable for a given cell type and independent of the glycosidase substrate in the EDIs or differences in functional glycosidase activity between cell lines. Measuring drug efflux of the immunostimulant payload and efflux protein expression revealed that EDI/cancer cell-mediated immunogenicity was governed by efflux potential of the cancer cells. We determined that, following EDI conversion, immunostimulant efflux occurred through both P-glycoprotein-dependent and P-glycoprotein-independent transport mechanisms. Overall, this study highlights the broad ability of EDIs to couple immunogenicity to the metabolism of many cancers that exhibit drug efflux and suggests that designing future generations of EDIs with immunostimulant payloads that are optimized for drug efflux could be particularly beneficial.
The tunable nature of phosphoramidate
linkers enables broad applicability
as pH-triggered controlled-release platforms, particularly in the
context of antibody- and small-molecule-drug conjugates (ADCs and
SMDCs), where there remains a need for new linker technology. Herein,
we explored in-depth the release of turn-on fluorogenic payloads from
a homoserinyl-based phosphoramidate acid-cleavable linker. Kinetics
of payload release from the scaffold was observed in buffers representing
the pH conditions of systemic circulation, early and late endosomes,
and lysosomes. It was found that payload release takes place in two
key consecutive steps: (1) P–N bond hydrolysis and (2) spacer
immolation. These two steps were found to follow pseudo-first-order
kinetics and had opposite dependencies on pH. P–N bond hydrolysis
increased with decreasing pH, while spacer immolation was most rapid
at physiological pH. Despite the contrasting release kinetics of these
two steps, maximal payload release was observed at the mildly acidic
pH (5.0–5.5), while minimal payload release occurred at physiological
pH. We integrated this phosphoramidate-payload linker system into
a PSMA-targeted fluorescent turn-on probe to study the intracellular
trafficking and release of a fluorescent payload in PSMA-expressing
prostate cancer cells. Results showed excellent turn-on and accumulation
of the coumarin payload in the late endosomal and lysosomal compartments
of these cells. The release properties of this linker mark it as an
attractive alternative in the modular design of ADCs and SMDCs, which
demand selective intracellular payload release triggered by the pH
changes that accompany intracellular trafficking.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.