Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Ultracompact 3D microfluidics for time-resolved structural biology

To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s−1, which allows for the use of megahertz XFEL repetition rates. By integrating an additional orifice, we implement a low consumption flow-focusing nozzle, which is validated by solving a hemoglobin structure. Also, aberration-free in operando X-ray microtomography is introduced to study efficient equivolumetric millisecond mixing in channels with 3D features integrated into the nozzle. Such devices can be printed in minutes by locally adjusting print resolution during fabrication. This technology has the potential to permit ultracompact devices and performance improvements through 3D flow optimization in all fields of microfluidic engineering

On the use of multilayer Laue lenses with X-ray Free Electron Lasers

Multilayer Laue lenses were used for the first time to focus x-rays from an X-ray Free Electron Laser (XFEL). In an experiment, which was performed at the European XFEL, we demonstrated focusing to a spot size of a few tens of nanometers. A series of runs in which the number of pulses per train was increased from 1 to 2, 3, 4, 5, 6, 7, 10, 20 and 30 pulses per train, all with a pulse separation of 3.55 us, was done using the same set of lenses. The increase in the number of pulses per train was accompanied with an increase of x-ray intensity (transmission) from 9% to 92% at 5 pulses per train, and then the transmission was reduced to 23.5 % when the pulses were increased further. The final working condition was 30 pulses per train and 23.5% transmission. Only at this condition we saw that the diffraction efficiency of the MLLs changed over the course of a pulse train, and this variation was reproducible from train to train. We present the procedure to align and characterize these lenses and discuss challenges working with the pulse trains from this unique x-ray source

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX

Inhibition of SARS-CoV-2 main protease by allosteric drug-binding

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to M$^{pro}$. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2