A Pharmacophore Model for SARS-CoV-2 3CLpro Small Molecule Inhibitors and in Vitro Experimental Validation of Computationally Screened Inhibitors

in Journal of Chemical Information and Modeling (2021), 61(8), 4082-4096

Among the biomedical efforts in response to the current coronavirus (COVID-19) pandemic, pharmacological strategies to reduce viral load in patients with severe forms of the disease are being studied ... [more ▼]

Among the biomedical efforts in response to the current coronavirus (COVID-19) pandemic, pharmacological strategies to reduce viral load in patients with severe forms of the disease are being studied intensively. One of the main drug target proteins proposed so far is the SARS-CoV-2 viral protease 3CLpro (also called Mpro), an essential component for viral replication. Ongoing ligand- and receptor-based computational screening efforts would be facilitated by an improved understanding of the electrostatic, hydrophobic and steric features that characterize small molecule inhibitors binding stably to 3CLpro, as well as by an extended collection of known binders. Here, we present combined virtual screening, molecular dynamics simulation, machine learning and in vitro experimental validation analyses which have led to the identification of small molecule inhibitors of 3CLpro with micromolar activity, and to a pharmacophore model that describes functional chemical groups associated with the molecular recognition of ligands by the 3CLpro binding pocket. Experimentally validated inhibitors using a ligand activity assay include natural compounds with available prior knowledge on safety and bioavailability properties, such as the natural compound rottlerin (IC50 = 37 µM), and synthetic compounds previously not characterized (e.g. compound CID 46897844, IC50 = 31 µM). In combination with the developed pharmacophore model, these and other confirmed 3CLpro inhibitors may provide a basis for further similarity-based screening in independent compound databases and structural design optimization efforts, to identify 3CLpro ligands with improved potency and selectivity. Overall, this study suggests that the integration of virtual screening, molecular dynamics simulations and machine learning can facilitate 3CLpro-targeted small molecule screening investigations. Different receptor-, ligand- and machine learning-based screening strategies provided complementary information, helping to increase the number and diversity of identified active compounds. Finally, the resulting pharmacophore model and experimentally validated small molecule inhibitors for 3CLpro provide resources to support follow-up computational screening efforts for this drug target. [less ▲]

PDE6D Inhibitors with a New Design Principle Selectively Block K‑Ras Activity

in ACS Omega (2020), 5(1), 832-842

High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors

in Analytical Biochemistry (2019), 572

The KRAS gene is highly mutated in human cancers and the focus of current Ras drug development efforts. Recently the interface between the C-terminus of K-Ras and calmodulin (CaM) was proposed as a target ... [more ▼]

The KRAS gene is highly mutated in human cancers and the focus of current Ras drug development efforts. Recently the interface between the C-terminus of K-Ras and calmodulin (CaM) was proposed as a target site to block K-Ras driven cancer cell stemness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel CaM-inhibitors as potential K-Ras stemness-signaling disruptors. A modulated time-resolved Förster resonance energy transfer (mTR-FRET)-assay was developed and benchmarked against an identically designed fluorescence anisotropy (FA)-assay. In both assays, two CaM-binding peptides were labeled with Eu(III)-chelate or fluorescein and used as single-label reporter probes that were displaced from CaM upon competitor binding. Thus, peptidic and small molecule competitors with nanomolar to micromolar affinities to CaM could be detected, including a peptide that was derived from the C-terminus of K-Ras. In order to detect CaM-residue specific covalent inhibitors, a cell lysate-based Förster resonance energy transfer (FRET)-assay was furthermore established. This assay enabled us to measure the slow, residue-specific, covalent inhibition by ophiobolin A in the presence of other endogenous proteins. In conclusion, we have developed a panel of fluorescence-assays that allows identification of conventional and covalent CaM-inhibitors as potential disruptors of K-Ras driven cancer cell stemness. [less ▲]

Combining chemical and genetic approaches for development of responsive FRET-based sensor systems for protein kinases

in Biophysical Chemistry (2016), 211

Bifunctional Ligands for Inhibition of Tight-Binding Protein–Protein Interactions

in Bioconjugate Chemistry (2016), 27(8), 1900-1910

FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases

in Analytical Biochemistry (2015)

Active site analysis of cis-epoxysuccinate hydrolase from Nocardia tartaricans using homology modeling and site-directed mutagenesis

in Applied Microbiology and Biotechnology (2012), 93(6), 23772386