Tag: fragment screening

50th Anniversary of the SSRL synchrotron radiation & protein crystallography initiative

50th Anniversary of the SSRL synchrotron radiation & protein crystallography initiative

Synchrotron-based protein crystallography continues to accelerate, driven by new and upgraded high-brightness sources, improved optics, faster large-area detectors, robust automation and streamlined data handling. These advances are making increasingly challenging structural biology projects feasible and are reshaping how synchrotron experiments integrate with today’s wider structural biology methods. While AI models are now routinely used in  molecular replacement software for macromolecular crystal structure determination, synchrotron experimental methods remain vital for detailed model refinement, and even validating AI models. Also extracting key chemical information, with anomalous dispersion at tuneable beamlines still playing an important role especially in identifying metals and other such atoms in proteins.

This special issue in Journal of Synchrotron Radiation, edited by John R. Helliwell and Marian Szebenyi, and their Overview with Colin Nave, with a Perspective from Keith Hodgson, as well as articles from a majority of the facilities worldwide, explores the evolving landscape in depth. It also highlights the expanding impact of fragment screening and binding studies (from cryogenic up to body temperatures) and the rapidly developing frontiers of time-resolved and serial crystallography. In particular, the issue charts the synergy between XFEL-based serial femtosecond crystallography and serial synchrotron crystallography, culminating in recent demonstrations of microsecond time resolution at upgraded synchrotrons such as ESRF–EBS, pointing to a future where synchrotrons and X-ray lasers together enable ever more powerful studies of biological structure, dynamics and function.

Access the special issue here

Image Credit:

Phillips, J.C., Wlodawer, A., Yevitz, M.M. and Hodgson, K.O., 1976. Applications of synchrotron radiation to protein crystallography: preliminary results. Proceedings of the National Academy of Sciences, 73(1), pp.128-132. 

Rosenbaum, G., Holmes, K.C. and Witz, J., 1971. Synchrotron radiation as a source for X-ray diffraction. Nature, 230(5294), pp.434-437.

Fast fragment discovery with protein crystals

Fast fragment discovery with protein crystals

Fragment-based drug discovery (FBDD) has become a standard approach for generating starting points in medicinal chemistry. Small fragments bind weakly but can be chemically elaborated into stronger ligands. The difficulty is that moving from weak binders to measurable leads usually involves multiple design-make-test-analyse (DMTA) cycles: each analogue must be synthesised, purified, tested biochemically, and crystallised. This is slow and leaves much chemical space unexplored.

Researchers developed a new technique called Binding-Site Purification of Actives (B-SPA) to bypass this bottleneck. Instead of purifying every product, they test crude reaction mixtures directly on protein crystals. Their study, published in Angewandte Chemie International Edition, shows how this works in practice. High-throughput macromolecular crystallography on Diamond’s I04-1 beamline enabled the team to detect which molecules bind, even if they were a minor product in a mixture. This structural “filtering” step dramatically accelerates hit-to-lead workflows.

Expanding fragments into thousands of analogues without purification

The team focused on the second bromodomain of PHIP ( disPHIP(2)), a protein implicated in epigenetic regulation and linked to cancers. A fragment hit (compound F709) had been identified in earlier crystallographic screens, but like most fragments, its binding was weak and undetectable in solution assays. Researchers wanted to explore chemical space around this initial fragment to see which modifications improve binding.

They designed up to six independent synthetic routes, each involving multi-step reactions (up to five synthetic steps), exploring different vectors of substitution (i.e. different parts of the fragment to substitute, such as replacing a ring, modifying substituents, adding functional groups). The designs were guided by synthetic tractability: only routes that are feasible with reliable chemistry were chosen. Using a low-cost robotic liquid handler, the group performed 1,876 reactions, generating diverse libraries of potential binders.

Each crude reaction mixture was checked by LC–MS using an automated tool (MSCheck) that flags the presence of the expected molecular ion. Out of 1,876 attempted syntheses, 1,108 mixtures (59%) contained the intended product. Rather than purify, the team directly soaked PHIP(2) crystals with these crude mixtures and collected data at Diamond’s I04-1 beamline, which is optimised for high-throughput macromolecular crystallography.

Read more on the Diamond website

Cell cytoskeleton as target for new active agents

Cell cytoskeleton as target for new active agents

Through a unique combination of computer simulations and laboratory experiments, researchers at the Paul Scherrer Institute PSI have discovered new binding sites for active agents – against cancer, for example – on a vital protein of the cell cytoskeleton. Eleven of the sites hadn’t been known before. The study is published in the journal Angewandte Chemie International Edition.

The protein tubulin is an essential building block of the so-called cell cytoskeleton. In cells, tubulin molecules arrange themselves into tube-like structures, the microtubule filaments. These give cells their shape, aid in transporting proteins and larger cellular components, and play a crucial role in cell division.

Thus tubulin performs diverse functions in the cell and in doing so interacts with numerous other substances. “Tubulin can bind an astonishing number of different proteins and small molecules, several hundred for sure,” says Tobias Mühlethaler, a doctoral candidate in the PSI Laboratory of Biomolecular Research and first author of the study. The functions of the protein are guided by means of such bonds. Also, many drugs dock on tubulin and take effect, for example, by preventing cell division in tumours.

Read more on the PSI website

Image: The research team in front of the Swiss Light Source (from left): Andrea Prota, Tobias Mühlethaler and Michel Steinmetz


Credit: Paul Scherrer Institute/Mahir Dzambegovic

Massive fragment screen points way to new SARS-CoV-2 inhibitors

Massive fragment screen points way to new SARS-CoV-2 inhibitors

Experiment with 2533 fragments compounds generates chemical map to future antiviral agents 

New research published in Science Advances provides a template for how to develop directly-acting antivirals with novel modes of action, that would combat COVID-19 by suppressing the SARS-CoV-2 viral infection. The study focused on the macrodomain part of the Nsp3 gene product that SARS-CoV-2 uses to suppress the host cell’s natural antiviral response. This part of the virus’s machinery, also known as Mac1, is essential for its reproduction: previous studies have shown that viruses that lack it cannot replicate in human cells, suggesting that blocking it with a drug would have the same effect.  

The study involved a crystallographic fragment screen of the Nsp3 Mac1 protein by an open science collaboration between researchers from the University of Oxford, the XChem platform at Diamond, and researchers from the QCRG Structural Biology Consortium at the University of California San Francisco.  The international effort discovered 234 fragment compounds that directly bind to sites of interest on the surface of the protein, and map out chemical motifs and protein-compound interactions that researchers and pharmaceutical companies can draw on to design compounds that could be developed into antiviral drugs.  This work is thus foundational for preparing for future pandemics.   

Read more on the Diamond website

Image: Principal Beamline Scientist on I04-1, Frank von Delft

Credit: Diamond Light Source