Unconventional approaches to kinase inhibition : covalent inhibitors and docking site inhibitors of mitogen-activated protein kinases

The body of work that follows is a description of biochemical screening methods to identify non-ATP competitive and covalent ERK inhibitors, as well as assay design for characterizing covalent inhibitors of JNK in an isoform-specific manner. A covalent ERK inhibitor is presented, as well as a novel class of non-covalent ERK docking site inhibitors. An assay to elucidate the kinetic parameters of covalent JNK inhibitors is described, and the kinetic parameters for the inhibitor JNK-IN-8 are illustrated as a case study. Additionally, a series of JNK-IN-8 analogues were tested in a PyVMT breast cancer cell model for their ability to preferentially inhibit either JNK1 or JNK2. The rationale behind targeting MAPKs at docking sites and focusing on covalent inhibitors is centered on several key elements. First, the proximity-based mechanism of MAPK catalysis depends on docking site interactions to direct substrate consensus sequences to the vicinity of the MAPK active site. MAPK activators, phosphatases, scaffolding proteins, and other molecules also engage docking sites. Therefore, targeting docking sites with inhibitors can not only directly or indirectly block substrate phosphorylation or enzyme activation, but the mechanisms of catalysis themselves can be altered. Secondly, the signal dynamics of MAPKs, such as their switch-like behavior, intra-pathway feedback, and inter-pathway crosstalk makes complete inhibition of MAPKs necessary to stop signaling in a therapeutic context. Covalent, irreversible MAPK inhibitors can induce complete inhibition by being effectively immune to substrate competition, and by forcing recovery of signaling to be fully dependent on either MAPK mutations, protein synthesis, or pathway bypass. Targeting a MAPK at multiple interaction sites can further promote a full blockade of signaling. The collective goal of the work presented in this dissertation is to contribute to the breadth of targeted kinase inhibitors that can be used against cancers, such as BRAF-V600E melanoma. These inhibitors and methods of inhibitor development can be used as tools to probe the functionality of individual protein-protein interaction sites on ERK and JNK, and the principles described here can be adapted to apply to other kinases and binding sites as needed in future studies.Biomedical Engineerin

The Craft Academy Robotics Project

To begin, the team’s research project consisted of a robot that could compete on a 12-foot by 12-foot playing field alongside 3 other robots. The robot had to fit within an 18in-by-18in limit, it could be made of a variety of materials and motors specified in the game manual to help it achieve the most amount of points possible. There are many ways to earn points in Freight Frenzy divided into 3 sections, Autonomous, Tele-Operation or “Op” for short, and End-Game: In the Autonomous section, the match starts with a 30-second Autonomous Period where Robots are operated only via pre-programmed instructions. Teams are not allowed to control Robot behavior with the Driver Station or any other actions. The Control Hub is placed in a hands-off location during said Autonomous Period so that it is evident that there is no human control of Robots. The robots can obtain points by parking in the Alliance storage unit, parking in the closest to the robot’s alliance, moving freight into the shipping hubs, or alliance specific storage unit, with an additional bonus of using a camera to recognize and move a rubber duck or Team Shipping Element to a randomized part of the shipping hub. During the Tele-Op part, teams can score points by going over or around the PVC barrier to obtain whiffle balls or different weighted plastic cubes and placing them in either the Alliance Shipping Hub or Shared Shipping Hub. Finally, in End-Game, Teams can put the custom capping element on the Alliance Shipping Hub, spin the Carrousel to get up to 10 ducks, or park in a Warehouse.https://scholarworks.moreheadstate.edu/celebration_posters_2022/1043/thumbnail.jp

A quantitative method to measure biofilm removal efficiency from complex biomaterial surfaces using SEM and image analysis

Biofilm accumulation on biomaterial surfaces is a major health concern and significant research efforts are directed towards producing biofilm resistant surfaces and developing biofilm removal techniques. To accurately evaluate biofilm growth and disruption on surfaces, accurate methods which give quantitative information on biofilm area are needed, as current methods are indirect and inaccurate. We demonstrate the use of machine learning algorithms to segment biofilm from scanning electron microscopy images. A case study showing disruption of biofilm from rough dental implant surfaces using cavitation bubbles from an ultrasonic scaler is used to validate the imaging and analysis protocol developed. Streptococcus mutans biofilm was disrupted from sandblasted, acid etched (SLA) Ti discs and polished Ti discs. Significant biofilm removal occurred due to cavitation from ultrasonic scaling (p < 0.001). The mean sensitivity and specificity values for segmentation of the SLA surface images were 0.80 ± 0.18 and 0.62 ± 0.20 respectively and 0.74 ± 0.13 and 0.86 ± 0.09 respectively for polished surfaces. Cavitation has potential to be used as a novel way to clean dental implants. This imaging and analysis method will be of value to other researchers and manufacturers wishing to study biofilm growth and removal