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Growing insect cells with high air space in culture vessel is common from the early development of suspension cell culture. We believed and followed it with the hope that it allows sufficient air for optimal cell growth. However, we missed to identify how much air exactly cells need for its growth and multiplication. Here we present the innovative method that changed the way we run insect cell culture. The method is easy to adapt, cost-effective and useful for both academic and industrial research labs. We believe this method will revolutionize the way we run insect cell culture by increasing throughput in a cost-effective way. In our study we identified: • Insect cells need to be in suspension; air space in culture vessel and type of culture vessel is of less importance. Shaking condition that introduces small air bubbles and maintains it in suspension for longer time provides better oxygen transfer in liquid. For this, high-fill volume in combination with speed and shaking diameter are important. • Commercially available insect cells are not fragile as original isolates. These cells can easily withstand higher shaking speed. • Growth condition in particular lab set-up needs to be optimized. The condition used in one lab may not be optimum for another lab due to different incubators from different vendors.

Phenotypic assays have become an established approach to drug discovery. Greater disease relevance is often achieved through cellular models with increased complexity and more detailed readouts, such as gene expression or advanced imaging. However, the intricate nature and cost of these assays impose limitations on their screening capacity, often restricting screens to well-characterized small compound sets such as chemogenomics libraries. Here, we outline a cheminformatics approach to identify a small set of compounds with likely novel mechanisms of action (MoAs), expanding the MoA search space for throughput limited phenotypic assays. Our approach is based on mining existing large-scale, phenotypic high-throughput screening (HTS) data. It enables the identification of chemotypes that exhibit selectivity across multiple cell-based assays, which are characterized by persistent and broad structure activity relationships (SAR). We validate the effectiveness of our approach in broad cellular profiling assays (Cell Painting, DRUG-seq, and Promotor Signature Profiling) and chemical proteomics experiments. These experiments revealed that the compounds behave similarly to known chemogenetic libraries, but with a notable bias toward novel protein targets. To foster collaboration and advance research in this area, we have curated a public set of such compounds based on the PubChem BioAssay dataset and made it available for use by the scientific community.

A series of inhibitors of Autotaxin (ATX) have been developed from a high throughput screening hit, 1a, which shows an alternative binding mode to known catalytic site inhibitors. Selectivity over the hERG channel and microsomal clearance were dependent on the lipophilicity of the compounds, and this was optimised by reduction of clogD whilst maintaining high affinity ATX inhibition. Compound 15a shows good oral exposure, and concentration dependent inhibition of formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic (PK/PD) experiments., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Baculovirus (BV) mediated insect cell expression system utilizes transfection as a first step to introduce recombinant baculovirus DNA into insect cells. Many labs are still relying on the conventional liposome based transfection method in adherent culture. Here we describe a more efficient method that can replace the existing method. This method is economical and does not require any special adjustment in existing labs. •An innovative method of transfecting insect cells in suspension using polyethyleneimine (PEI) is described here.•The beauty of this method is minimal intermediate manipulation of culture during transfection and virus generation.•The method significantly reduces the chances of cross contamination of viruses while handling multiple targets and constructs as well as the other microbial contamination.

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen-containing bisphosphonate (N-BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N-BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS-N-BP complexes rather than to compete with and displace the N-BP inhibitor. Stabilization of the FPPS-N-BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N-BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N-BP drugs. Moreover, our data form the basis for structure-guided design of optimized N-BPs with improved pharmacological properties.