(L-479) Understanding the effect of Genetic Variants on Oxytocin-Oxytocin Receptor Binding: A Mathematical Model

Background/Aim: Oxytocin (OXT) is administered to approximately two million pregnant women annually in the USA to augment uterine contractions during labor and delivery. However, there is wide variability in doses used due to variations in individual response. We recently described five oxytocin receptor (OXTR) genetic variants, two of which have reduced plasma membrane localization and OXT-induced signaling compared to wild type OXTR. Thus, we sought to determine the impact of reduced cell surface OXTR localization on the OXT:OXTR binding using a data-driven mathematical and computational model.
Materials and Methods: We constructed a mathematical model to elucidate the OXT:OXTR binding of wildtype OXTR and five OXTR variants (V281M, E339K, V45L, P108A, L206V), using ordinary differential equations. We parameterized the model via an extensive literature mining of published data on the equilibrium dissociation constant (Kd, binding affinity) for myometrial smooth muscle cells and OXTR transfected HEK293T cells (Figure 1). We used the following OXT:OXTR association (kon) and dissociation (koff) rate constants available in the literature: kon = 6.8 x10-4 nM-1min-1 and koff = 0.0011 min-1 (Kd = 1.6 nM) for human myometrial cells and kon = 8.8 x10-3 nM-1min-1 and koff = 0.005 min-1 (Kd = 0.56 nM) for OXTR transfected HEK293T cells. Initial concentrations of OXTR and OXTR variants from recently published work from our lab [1]: [wt] = 1.68 nM, [V281M] = 0.75 nM, [P108A] = 2.66 nM, [L206V] = 3.04 nM, [V45L] = 1.96 nM, [E339K] = 1.19 nM [2] and [OXT] = 1000 nM. The equilibrium is defined as the maximal OXT:OXTR binding. We performed simulations using SciPy, NumPy, and Matplotlib libraries of python 3.9.7.

Results and Discussions: We compared equilibrium OXT:OXTR dynamics when OXTR was transfected into a common heterologous expression cell line, HEK293Ts, versus transfected into a myometrial cell line. OXT:OXTR complex equilibrium was achieved much slower in myometrial cells (~ 10 mins) compared to OXTR transfected HEK293T cells (~ 1 min), as was expected by the 3-fold difference in Kd. Next, we examined OXT:OXTR complex equilibrium for all genetic variants in both cell types. OXT:OXTR complex equilibria are significantly diminished in myometrial cells, 55% and 29% lower than WT, in V281M and E339K, respectively (Figure 2). Thus, predicting that myometrial cells with V281M may require 4.5-fold higher OXT than WT to achieve activation level in 45 seconds, and E339K may require 2.5-fold higher OXT than WT to achieve activation level in 90 seconds. To increase complex formation of the V281M and E339K OXTR variants, we tested binding affinity enhancement. V281M achieved WT complex formation in 1 min with only 1000 nM OXT if the Kd shifted to 0.05 nM, representing at least a 3-fold increase in the kon or 9-fold decrease in the koff. Similarly, E339K could achieve WT levels of complex formation in 90 seconds if the Kd is lowered to 0.08 nM, requiring either a 2-fold increase in kon or 11-fold decrease in koff .

Conclusions: Our data-driven mathematical and computational model provides a novel platform for understanding how genetic variants of OXTR affect OXT response. The V281M and E339K mutations (0.3% of gnomAD participants [1]) may decrease OXT activation and signaling. We show that the V281M mutation requires a 4.5-fold higher OXT dose, which raises safety and feasibility concerns for OXT dosing in patients carrying this mutation. Thus, our approach can inform clinical decisions with potential for further patient-specific parameterization. Moreover, our platform offers a unique opportunity to compare in vivo human pregnancy with in vitro systems like HEK293T cells transfected with OXTR and even preclinical animal models, see Figure 1. Future investigations will incorporate OXTR desensitization and plasma membrane trafficking to identify additional therapeutic targets for rescuing wild-type function across patients. Overall, our approach, using biomedical engineering principles, is leading the way to the safe and effective use of oxytocin in labor and delivery.

Acknowledgement: The research reported in this publication was supported by the
Eunice Kennedy Shriver National Institute of Child Health and Human Development
under Award Number 5R01HD096737-04. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References:
[1] M. Malik et al., “Naturally Occurring Genetic Variants in the Oxytocin Receptor Alter Receptor Signaling Profiles,” ACS Pharmacol Transl Sci, vol. 4, no. 5, pp. 1543–1555, Oct. 2021, doi: 10.1021/acsptsci.1c00095.
[2] S. Phaneuf et al., “The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA,” 1997.