Correction to "Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity".

[This corrects the article DOI: 10.1021/acsptsci.3c00313.].

Development of Highly Potent and Selective Covalent FGFR4 Inhibitors Based on SNAr Electrophiles.

Fibroblast growth factor receptor 4 (FGFR4) is thought to be a driver in several cancer types, most notably in hepatocellular carcinoma. One way to achieve high potency and isoform selectivity for FGFR4 is covalently targeting a rare cysteine (C552) in the hinge region of its kinase domain that is not present in other FGFR family members (FGFR1-3). Typically, this cysteine is addressed via classical acrylamide electrophiles. We demonstrate that noncanonical covalent "warheads" based on nucleophilic aromatic substitution (SNAr) chemistry can be employed in a rational manner to generate highly potent and (isoform-)selective FGFR4 inhibitors with a low intrinsic reactivity. Key compounds showed low to subnanomolar potency, efficient covalent inactivation kinetics, and excellent selectivity against the other FGFRs, the kinases with an equivalent cysteine, and a representative subset of the kinome. Moreover, these compounds achieved nanomolar potencies in cellular assays and demonstrated good microsomal stability, highlighting the potential of SNAr-based approaches in covalent inhibitor design.

Cathepsin-Targeting SARS-CoV-2 Inhibitors: Design, Synthesis, and Biological Activity.

Cathepsins (Cats) are proteases that mediate the successful entry of SARS-CoV-2 into host cells. We designed and synthesized a tailored series of 21 peptidomimetics and evaluated their inhibitory activity against human cathepsins L, B, and S. Structural diversity was realized by combinations of different C-terminal warhead functions and N-terminal capping groups, while a central Leu-Phe fragment was maintained. Several compounds were identified as promising cathepsin L and S inhibitors with Ki values in the low nanomolar to subnanomolar range, for example, the peptide aldehydes 9a and 9b (9a, 2.67 nM, CatL; 0.455 nM, CatS; 9b, 1.76 nM, CatL; 0.512 nM, CatS). The compounds' inhibitory activity against the main protease of SARS-CoV-2 (Mpro) was additionally investigated. Based on the results at CatL, CatS, and Mpro, selected inhibitors were subjected to investigations of their antiviral activity in cell-based assays. In particular, the peptide nitrile 11e exhibited promising antiviral activity with an EC50 value of 38.4 nM in Calu-3 cells without showing cytotoxicity. High metabolic stability and favorable pharmacokinetic properties make 11e suitable for further preclinical development.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

Development of Ligands for the Super Conserved Orphan G Protein-Coupled Receptor GPR27 with Improved Efficacy and Potency.

The orphan G protein-coupled receptor GPR27 appears to play a role in insulin production, secretion, lipid metabolism, neuronal plasticity, and l-lactate homeostasis. However, investigations on the function of GPR27 are impaired by the lack of potent and efficacious agonists. We describe herein the development of di- and trisubstituted benzamide derivatives 4a-e, 7a-z, and 7aa-ai, which display GPR27-specific activity in a ß-arrestin 2 recruitment-based assay. Highlighted compounds are PT-91 (7p: pEC50 6.15; Emax 100%) and 7ab (pEC50 6.56; Emax 99%). A putative binding mode was revealed by the docking studies of 7p and 7ab with a GPR27 homology model. The novel active compounds exhibited no GPR27-mediated activation of G proteins, indicating that the receptor may possess an atypical profile. Compound 7p displays high metabolic stability and brain exposure in mice. Thus, 7p represents a novel tool to investigate the elusive pharmacology of GPR27 and assess its potential as a drug target.

Drivers passing cyclists: How does sight distance affect safety? Results from a naturalistic study.

INTRODUCTION: Cycling is popular for its ecological, economic, and health benefits. However, especially in rural areas, cyclists may need to share the road with motorized traffic, which is often perceived as a threat. Overtaking a cyclist is a particularly critical maneuver for drivers as they need to control their lateral clearance and speed when passing the cyclist, possibly in the presence of oncoming vehicles or view-obstructing curves. An overtaking vehicle can destabilize the cyclist when passing with low clearance and high speed. At the same time, the cyclist may get scared and eventually stop cycling. In this work, we investigated how visibility regarding available sight distance-an important factor for infrastructure design and regulation-affects drivers' behavior when overtaking cyclists. METHOD: Using four roadside-based traffic sensors, we collected naturalistic data that contained kinematics of drivers overtaking cyclists on a rural road in Sweden. We modeled lateral clearance and speed at the passing moment in response to variables such as sight distance and oncoming traffic with a Bayesian multivariate approach. RESULTS: Fitted on 81 maneuvers, the model revealed that drivers reduced lateral clearance under reduced sight distance. Speed was similarly reduced, however, not as clearly. When an oncoming vehicle was present, it had a similar-yet stronger-effect than sight distance. While we found an overall correlation between clearance and speed, some maneuvers were recorded at critically low clearance. CONCLUSIONS: Cyclists' safety is endangered when passed by drivers under reduced visibility or close to oncoming traffic. PRACTICAL APPLICATIONS: Decision-making for infrastructure and policymaking should aim at prohibiting overtaking in areas with reduced visibility or close oncoming traffic. The model developed in this study may serve as a reference to vehicle active-safety systems and automated driving. The collected and processed data may support evaluating driver models fitted on less ecologically valid data and simulated active-safety systems.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors.

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The reengineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

How do different micro-mobility vehicles affect longitudinal control? Results from a field experiment.

INTRODUCTION: While micromobility vehicles offer new transport opportunities and may decrease fuel emissions, the extent to which these benefits outweigh the safety costs is still uncertain. For instance, e-scooterists have been reported to experience a tenfold crash risk compared to ordinary cyclists. Today, we still do not know whether the real safety problem is the vehicle, the human, or the infrastructure. In other words, the new vehicles may not necessarily be unsafe; the behavior of their riders, in combination with an infrastructure that was not designed to accommodate micromobility, may be the real issue. METHOD: In this paper, we compared e-scooters and Segways with bicycles in field trials to determine whether these new vehicles create different constraints for longitudinal control (e.g., in braking avoidance maneuvers). RESULTS: The results show that acceleration and deceleration performance changes across vehicles; specifically, e-scooters and Segways that we tested cannot brake as efficiently as bicycles. Further, bicycles are experienced as more stable, maneuverable, and safe than Segways and e-scooters. We also derived kinematic models for acceleration and braking that can be used to predict rider trajectories in active safety systems. PRACTICAL APPLICATIONS: The results from this study suggest that, while new micromobility solutions may not be intrinsically unsafe, they may require some behavior and/or infrastructure adaptations to improve their safety. We also discuss how policy making, safety system design, and traffic education may use our results to support the safe integration of micromobility into the transport system.

A data-driven framework for the safe integration of micro-mobility into the transport system: Comparing bicycles and e-scooters in field trials.

INTRODUCTION: Recent advances in technology create new opportunities for micro-mobility solutions even as they pose new challenges to transport safety. For instance, in the last few years, e-scooters have become increasingly popular in several cities worldwide; however, in many cases, the municipalities were simply unprepared for the new competition for urban space between traditional road users and e-scooters, so that bans became a necessary, albeit drastic, solution. In many countries, traditional vehicles (such as bicycles) may not be intrinsically safer than e-scooters but are considered less of a safety threat, possibly because-for cyclists-social norms, traffic regulations, and access to infrastructure are established, reducing the number of negative stakeholders. Understanding e-scooter kinematics and e-scooterist behavior may help resolve conflicts among road users, by favoring a data-driven integration of these new e-vehicles into the transport system. In fact, regulations and solutions supported by data are more likely to be acceptable and effective for all stakeholders. As new personal-mobility solutions enter the market, e-scooters may just be the beginning of a micro-mobility revolution. METHOD: This paper introduces a framework (including planning, execution, analysis, and modeling) for a data-driven evaluation of micro-mobility vehicles. The framework leverages our experience assessing bicycle dynamics in real traffic to make objective and subjective comparisons across different micro-mobility solutions. In this paper, we use the framework to compare bicycles and e-scooters in field tests. RESULTS: The preliminary results show that e-scooters may be more maneuverable and comfortable than bicycles, although the former require longer braking distances. PRACTICAL APPLICATIONS: Data collected from e-scooters may, in the short term, facilitate policy making, geo-fencing solutions, and education; in the long run, the same data will promote the integration of e-scooters into a cooperative transport system in which connected automated vehicles share the urban space with micro-mobility vehicles. Finally, the framework and the models presented in this paper may serve as a reference for the future assessment of new micro-mobility vehicles and their users' behavior (although advances in technology and novel micro-mobility solutions will inevitably require some adjustments).

Modelling duration of car-bicycles overtaking manoeuvres on two-lane rural roads using naturalistic data.

Nowadays, Spanish two-lane rural roads frequently accommodate sport cyclists. They usually ride on the shoulder or on the right edge of the lane, sharing the infrastructure with motorised vehicles. Due to the speed difference between road users, the most frequent and dangerous interaction is in overtaking manoeuvres. One key factor from a safety and traffic operation point of view is the overtaking duration. The main aim of this paper is to analyse how factors related to the road, the cyclists, and the overtaking manoeuvre influence the duration of overtaking to cyclists on two-lane rural roads. Naturalistic field data were obtained using instrumented bicycles. Seven groups of cyclists, formed by different numbers of cyclists riding in-line and two-abreast, rode along five rural roads with different geometric and traffic characteristics. A total of 1592 flying manoeuvres, in which drivers did not reduce their speed, and 192 accelerative manoeuvres were analysed. The overtaking duration, considering each overtaking strategy, was modelled using Bayesian statistics. Results showed that flying manoeuvres were more prevalent than accelerative. They were performed with higher speeds and lower lateral clearances and, therefore, presented lower overtaking durations. For both overtaking strategies, duration increased on wider roads and with a larger size of the group. The presence of an oncoming vehicle decreased the overtaking duration. However, other factors presented opposite effects on the duration depending on the overtaking strategy. The developed predictive models allow obtaining overtaking durations varying road and cyclist grouping characteristics. Results can be used by road administration to manage and propose some specific countermeasures to integrate the cyclists in a safe and efficient way on two-lane rural roads.

Modelling cyclists' comfort zones from obstacle avoidance manoeuvres.

This paper introduces a framework for modelling the cyclist's comfort zone. Unlike the driver's comfort zone, little is known about the cyclist's. The framework draws on existing literature in cognitive science about driver behaviour to explain experimental results from cycling field trials, and the modelling of these results. We modelled braking and steering manoeuvres from field data of cyclists' obstacle avoidance within their comfort zone. Results show that when cyclists avoided obstacles by braking, they kept a constant deceleration; as speed increased, they started to brake earlier, farther from the obstacle, maintaining an almost constant time to collision. When cyclists avoided obstacles by steering, they maintained a constant distance from the object, independent of speed. Overall, the higher the speed, the more the steering manoeuvres were temporally delayed compared to braking manoeuvres. We discuss these results and other similarities between cyclist and driver behaviour during obstacle avoidance. Implications for the design of acceptable active safety and infrastructure design are also addressed.

How do oncoming traffic and cyclist lane position influence cyclist overtaking by drivers?

Overtaking cyclists is challenging for drivers because it requires a well-timed, safe interaction between the driver, the cyclist, and the oncoming traffic. Previous research has investigated this manoeuvre in different experimental environments, including naturalistic driving, naturalistic cycling, and simulator studies. These studies highlight the significance of oncoming traffic-but did not extensively examine the influence of the cyclist's position within the lane. In this study, we performed a test-track experiment to investigate how oncoming traffic and position of the cyclist within the lane influence overtaking. Participants overtook a robot cyclist, which was controlled to ride in two different lateral positions within the lane. At the same time, an oncoming robot vehicle was controlled to meet the participant's vehicle with either 6 or 9 s time-to-collision. The order of scenarios was randomised over participants. We analysed safety metrics for the four different overtaking phases, reflecting drivers' safety margins to rear-end, head-on, and side-swipe collisions, in order to investigate the two binary factors: 1) time gap between ego vehicle and oncoming vehicle, and 2) cyclist lateral position. Finally, the effects of these two factors on the safety metrics and the overtaking strategy (either flying or accelerative depending on whether the overtaking happened before or after the oncoming vehicle had passed) were analysed. The results showed that, both when the cyclist rode closer to the centre of the lane and when the time gap to the oncoming vehicle was shorter, safety margins for all potential collisions decreased. Under these conditions, drivers-particularly female drivers-preferred accelerative over flying manoeuvres. Bayesian statistics modelled these results to inform the development of active safety systems that can support drivers in safely overtaking cyclists.

How do drivers overtake pedestrians? Evidence from field test and naturalistic driving data.

For pedestrians, the risk of dying in a traffic accident is highest on rural roads, which are often characterized by a lack of sidewalks and high traffic speed. In fact, hitting the pedestrian during an overtaking attempt is a common crash scenario. To develop active safety systems that avoid such crashes, it is necessary to understand and model driver behavior during the overtaking maneuvers, so that system interventions are acceptable because they happen outside drivers' comfort zone. Previous modeling of driver behavior in interactions with pedestrians primarily focused on road crossing scenarios. The aim of this study was, instead, to address pedestrian-overtaking maneuvers on rural roads. We focused our analysis on how drivers adjust their behavior with respect to three safety metrics (in order of importance): 1) minimum lateral clearance when passing the pedestrian, 2) overtaking speed at that moment, and 3) the time-to-collision at the moment of steering away to start the overtaking maneuver. The influence of three factors on the safety metrics was investigated: 1) walking direction (same as the overtaking vehicle or opposite), 2) walking position (on the edge of the vehicle lane or 0.5 m away from the edge on the paved shoulder), and 3) oncoming traffic (absent or present). Seventy-seven overtaking maneuvers in France from the naturalistic driving study UDRIVE and 297 maneuvers in Sweden from field tests were analyzed. Bayesian regression was used to model how minimum lateral clearance and overtaking speed depended on the three factors. Results showed that drivers maintained smaller minimum lateral clearance and lower overtaking speed when the pedestrian was walking in the opposite direction, on the lane edge, or when oncoming traffic was present. Minimum lateral clearance and time-to-collision were only weakly correlated with overtaking speed. The regression models predicted distributions similar to those actually observed in the data. The time-to-collision at the moment of steering away was comparable in value to the time-to-collision used by Euro NCAP for testing active safety systems in car-to-pedestrian longitudinal scenarios since 2018. This study is the first to analyze driver behavior when overtaking pedestrians, based on field test and naturalistic driving data. Results suggest that pedestrian safety is particularly endangered in situations when the pedestrian is walking opposite to traffic, close to the lane, and when oncoming traffic is present. The Bayesian regression models from this study can be used in active safety systems to model drivers' comfort in overtaking maneuvers.

An Ex Vivo Bone Defect Model to Evaluate Bone Substitutes and Associated Bone Regeneration Processes.

The increased incidence of bone defects, especially in cases of comminuted fractures or bone tumor resections demands suitable bone grafts and substitutes. The aim of this study was to establish an ex vivo bone defect model to evaluate new bone substitutes and associated repair processes under controlled conditions. Femoral heads derived from patients undergoing total hip replacement were cut into cylinders (20 mm diameter, 7 mm height). A central bone defect (6 mm diameter, 5 mm depth) was inserted centrally. The bone slides were cultured for 28 days and viability was evaluated by lactate dehydrogenase and alkaline phosphatase assay, and Calcein-AM viability staining and DNA quantification. Data revealed the viability of the bone tissue over the tested time period of 28 days, and an increase in cell numbers implicating active cell proliferation processes in the sections. To analyze the bone regeneration potential of this model in combination with a bone replacement material, we injected a collagen-type 1 hydrogel into the central defect. Cellular ingrowth into the gel was evaluated by microscopy and DNA quantification at different time points demonstrating an increase of cells in the defect over time. Finally, gene expression of osteogenic markers indicated an osteoblastic phenotype of the cells in the defect. In summary, the ex vivo bone defect model remains viable and shows active bone repair processes over 28 days. Additional advantages include high reproducibility, manageable costs, and a native bone-implant interface supporting the evaluation of bone substitute materials and associated regeneration processes. Impact statement Testing of new implant materials and bone repair strategies up to date rely mainly on in vivo and in vitro investigation models providing different pros and cons. In this study we established a novel human ex vivo bone defect model with a proven vitality of at least 28 days. The model provides a native bone implant interface and is designed to monitor cell invasion into a critically sized defect filled with the potential implant material. Furthermore, associated repair processes can be documented on the cell and molecular level, including additional advantages such as high reproducibility and manageable costs.

Evaluation of bone allograft processing methods: Impact on decellularization efficacy, biocompatibility and mesenchymal stem cell functionality.

In an ever-aging society the demand for bone-defect filling grafts continues to gain in importance. While autologous grafting still prevails as the gold standard, allografts and xenografts present viable alternatives with promising results. Physiochemical properties of a graft strongly depend on the processing method such as the decellularization protocol. In addition, the physiochemical characteristics are critical factors for a successful integration of the graft after the implantation and might influence mesenchymal stem cell function in therapeutic approaches combining grafts and autologous mesenchymal stem cells (MSCs). Several decellularization methods have been proposed, however it still remains unclear which method results in favorable physiochemical properties or might be preferred in stem cell applications. In the first part of this study we compared two decellularization approaches resulting in chemically processed allografts (CPAs) or sonication-based processed allografts (SPAs). Each decellularization approach was compared for its decellularization efficacy and its influence on the grafts' surface texture and composition. In the second part of this study biocompatibility of grafts was assessed by testing the effect of extraction medium on MSC viability and comparing them to commercially available allografts and xenografts. Additionally, grafts' performance in terms of MSC functionality was assessed by reseeding with MSCs pre-differentiated in osteogenic medium and determining cell adhesion, proliferation, as well as alkaline phosphatase (ALP) activity and the degree of mineralization. In summary, results indicate a more effective decellularization for the SPA approach in comparison to the CPA approach. Even though SPA extracts induced a decrease in MSC viability, MSC performance after reseeding was comparable to commercially available grafts based on DNA quantification, alkaline phosphatase activity and quantification of mineralization. Commercial Tutoplast allografts showed overall the best effects on MSC functionality as indicated by extraction biocompatibility testing as well as by comparing proliferation and osteogenic differentiation.