Fragment-Based Screen of SARS-CoV-2 Papain-like Protease (PLpro).

Coronaviruses have been responsible for numerous viral outbreaks in the past two decades due to the high transmission rate of this family of viruses. The deadliest outbreak is the recent Covid-19 pandemic, which resulted in over 7 million deaths worldwide. SARS-CoV-2 papain-like protease (PLPro) plays a key role in both viral replication and host immune suppression and is highly conserved across the coronavirus family, making it an ideal drug target. Herein we describe a fragment-based screen against PLPro using protein-observed NMR experiments, identifying 77 hit fragments. Analyses of NMR perturbation patterns and X-ray cocrystallized structures reveal fragments bind to two distinct regions of the protein. Importantly none of the fragments identified belong to the same chemical class as the few reported inhibitors, allowing for the discovery of a novel class of PLPro inhibitors.

Structure-Based Discovery of Potent, Orally Bioavailable Benzoxazepinone-Based WD Repeat Domain 5 Inhibitors.

The chromatin-associated protein WDR5 (WD repeat domain 5) is an essential cofactor for MYC and a conserved regulator of ribosome protein gene transcription. It is also a high-profile target for anti-cancer drug discovery, with proposed utility against both solid and hematological malignancies. We have previously discovered potent dihydroisoquinolinone-based WDR5 WIN-site inhibitors with demonstrated efficacy and safety in animal models. In this study, we sought to optimize the bicyclic core to discover a novel series of WDR5 WIN-site inhibitors with improved potency and physicochemical properties. We identified the 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one core as an alternative scaffold for potent WDR5 inhibitors. Additionally, we used X-ray structural analysis to design partially saturated bicyclic P7 units. These benzoxazepinone-based inhibitors exhibited increased cellular potency and selectivity and favorable physicochemical properties compared to our best-in-class dihydroisoquinolinone-based counterparts. This study opens avenues to discover more advanced WDR5 WIN-site inhibitors and supports their development as novel anti-cancer therapeutics.

Introducing Wound Healing Assays in the Undergraduate Biology Laboratory Using Ibidi Plates.

The wound healing assay is a simple and inexpensive method that allows researchers to experimentally mimic cell growth and migration leading to wound healing. In this assay, a wound is created on a monolayer of cultured mammalian cells and cell migration is monitored. Micrographs are captured at regular intervals during the duration of the experiment. These microscopy images are analyzed to compare cell migration and wound closure under different conditions. Introduction of different cytotoxic treatments into a wound healing assay can provide information as to whether a particular drug or compound of interest has the ability to affect cell migration. This type of analysis is important when assessing the ability of a particular cancer cell line to display invasive and metastatic behaviors. One of the challenges of this assay is to create the original wound in a way that is consistent across plates or treatments, facilitating comparisons across experimental groups. This is a particular challenge when using the wound healing assay in the context of an undergraduate biology class to expose students to a distinct form of mammalian cell culture and help them apply scientific knowledge and research skills. We found an easy way to overcome this obstacle by using ibidi plates. In this article, we provide a simple protocol to use ibidi plates and HeLa cells to set up wound healing assays. This laboratory exercise allows undergraduate students to utilize different skills developed through cell culture experience, such as growing, treating, and imaging mammalian cells.

Cryopreservation and the Freeze-Thaw Stress Response in Yeast.

The ability of yeast to survive freezing and thawing is most frequently considered in the context of cryopreservation, a practical step in both industrial and research applications of these organisms. However, it also relates to an evolved ability to withstand freeze-thaw stress that is integrated with a larger network of survival responses. These responses vary between different strains and species of yeast according to the environments to which they are adapted, and the basis of this adaptation appears to be both conditioned and genetic in origin. This review article briefly touches upon common yeast cryopreservation methods and describes in detail what is known about the biochemical and genetic determinants of cell viability following freeze-thaw stress. While we focus on the budding yeast Saccharomyces cerevisiae, in which the freeze-thaw stress response is best understood, we also highlight the emerging diversity of yeast freeze-thaw responses as a manifestation of biodiversity among these organisms.

Using The Cancer Genome Atlas as an Inquiry Tool in the Undergraduate Classroom.

Undergraduate students in the biomedical sciences are often interested in future health-focused careers. This presents opportunities for instructors in genetics, molecular biology, and cancer biology to capture their attention using lab experiences built around clinically relevant data. As biomedical science in general becomes increasingly dependent on high-throughput data, well-established scientific databases such as The Cancer Genome Atlas (TCGA) have become publicly available tools for medically relevant inquiry. The best feature of this database is that it bridges the molecular features of cancer to human clinical outcomes-allowing students to see a direct connection between the molecular sciences and their future professions. We have developed and tested a learning module that leverages the power of TCGA datasets to engage students to use the data to generate and test hypotheses and to apply statistical tests to evaluate significance.