Binding Position Dependent Modulation of Smoothened Activity by Cyclopamine.

Cyclopamine is a natural alkaloid that is known to act as an agonist when it binds to the Cysteine Rich Domain (CRD) of the Smoothened receptor and as an antagonist when it binds to the Transmembrane Domain (TMD). To study the effect of cyclopamine binding to each binding site experimentally, mutations in the other site are required. Hence, simulations are critical for understanding the WT activity due to binding at different sites. Additionally, there is a possibility that cyclopamine could bind to both sites simultaneously especially at high concentration, the implications of which remain unknown. We performed three independent sets of simulations to observe the receptor activation with cyclopamine bound to each site independently (CRD, TMD) and bound to both sites simultaneously. Using multi-milliseconds long aggregate MD simulations combined with Markov state models and machine learning, we explored the dynamic behavior of cyclopamine's interactions with different domains of WT SMO. A higher population of the active state at equilibrium, a lower activation free energy barrier of ~ 2 kcal/mol, and expansion of the hydrophobic tunnel to facilitate cholesterol transport agrees with the cyclopamine's agonistic behavior when bound to the CRD of SMO. A higher population of the inactive state at equilibrium, a higher free energy barrier of ~ 4 kcal/mol and restricted the hydrophobic tunnel to impede cholesterol transport showed cyclopamine's antagonistic behavior when bound to TMD. With cyclopamine bound to both sites, there was a slightly larger inactive population at equilibrium and an increased free energy barrier (~ 3.5 kcal/mol). The tunnel was slightly larger than when solely bound to TMD, and showed a balance between agonism and antagonism with respect to residue movements exhibiting an overall weak antagonistic effect.

Getting it right with discrete choice experiments: Are we hot or cold?

Discrete Choice Experiments (DCEs) are widely employed survey-based methods to assess preferences for healthcare services and products. While they offer an experimental way to represent health-related decisions, the stylized representation of scenarios in DCEs may overlook contextual factors that could influence decision-making. The aim of this paper was to evaluate the predictive validity of preferences elicited through a DCE in decisions likely influenced by a hot-cold empathy gap, and compare it to another commonly used method, a direct-elicitation question. We focused on preferences for pain-relief modalities, especially for an epidural during childbirth - a context where direct-elicitation questions have shown a preference for or intention to have a natural birth (representing the "cold" state), yet individuals often opt for an epidural during labor (representing the "hot" state). Leveraging a unique dataset collected from 248 individuals, we incorporated both the stated preferences collected through a survey administered upon hospital admission for childbirth and the actual pain-relief modality usage data documented in medical records. The DCE allowed for the evaluation of scenarios outside of those expected by respondents to simulate decision-making during childbirth. When we compared the predicted epidural use with the actual epidural use during labor, we observed a choice concordance of 71-60%, depending on the model specification. The concordance rate between the predicted and actual choices increased to 77-76% when accounting for the initial use of other ineffective modalities. In contrast, the direct-elicitation choices, relying solely on respondents' baseline expectations, yielded a lower concordance rate of 58% with actual epidural use. These findings highlight the flexibility of the DCE method in simulating complex decision contexts, including those involving hot-cold empathy gaps. The DCE proves valuable in assessing nuanced preferences, providing a more accurate representation of the decision-making processes in healthcare scenarios.