Protein X-ray Crystallography Research Articles

Development of a DNA damage response (DDR) therapeutics platform for oncology.

e15036 Background: Cancer cells respond to increases in DNA damage by deploying their DNA damage response (DDR) pathways. We are building a platform for the discovery and development of target-specific DDR therapeutics, including small molecule inhibitors and targeted protein degradation warheads, founded on fragment- and structure-based drug discovery. Methods: Our DDR platform, which includes hit-to-lead, lead optimization and candidate selection, starts with hit generation from a new technology that uses high-throughput protein X-ray crystallography to directly screen compound libraries. Our hit generation process produces empirical evidence of direct target engagement. The elucidation of high-quality ligand-bound 3D structures reveals the location and pose of the ligand and details of the protein-ligand interactions. Thus we can predict the structure-function consequences of the hit molecule engagement, which sets the stage for rapid assessment of synthetic tractability and intellectual property. After hit identification, we apply a multi-pronged approach in hit-to-lead conversion and lead optimization using iterative biophysical and biochemical assays, coupled with crystallography. We are applying our approach to several new targets in DDR and will present some early progress in this space. Results: Apurinic/apyrimidinic endonuclease 1 (APE1) is the major repair enzyme for abasic sites in DNA and contributes to DNA strand break processing. Many studies have associated increased APE1 levels with enhanced growth, migration, and drug resistance in human tumor cells, and with decreased patient survival. APE1 has been implicated in over 20 human cancers, including glioblastoma, making the protein an attractive target for the development of anticancer therapeutics. Despite intensive effort, there are no clinical endonuclease inhibitors of APE1. We have identified 25 diverse small molecule fragments that bind to APE1 at two distinct sites, including the endonuclease site. Pol eta (or PolH) is a DNA polymerase implicated, among other things, in the development of cisplatin resistance in a subset of ovarian cancers. In our quest to develop PolH inhibitors, we have identified 5 diverse fragments that bind to two distinct sites in the polymerase including a new potential allosteric site. Our results on APE1 and PolH represent the first known cases of crystal structures of small molecules bound to these proteins. Flap endonuclease (FEN1) is implicated in several cancers including for example ER/tamoxifen-resistant breast cancer. We are developing a targeted protein degradation approach using PROTACs (Proteolysis-Targeting Chimeras) toward the development of novel therapeutics against FEN1. Conclusions: Our results will help us develop small molecule inhibitors and targeted protein degradation against DDR targets that may be effective as single therapies or be used to make existing therapies more effective.

The Glitazone Class of Drugs as Carbonic Anhydrase Inhibitors-A Spin-Off Discovery from Fragment Screening.

The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen–deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

Profile of James H. Hurley

James “Jim” Hurley’s first introduction to crystal structures occurred when he was just 8 years old. While living north of Boulder, Colorado, his mother would take him to look for minerals near old mines in the mountains. “I learned the seven crystal systems at that age as part of learning how to identify the minerals that I found, and that came in handy later on when I did X-ray crystallography of proteins,” he says. James H. Hurley. Image credit: Calin Hurley (photographer). Hurley’s early fascination with minerals led him to a tentative interest in geology, but in college he decided to study physics instead. “As an undergraduate, I fell in love with the elegance of physics, and I was also fascinated by the complexity of life,” says Hurley. To combine those interests, Hurley pursued a doctorate in biophysics. “I thought there must be some way you could put the elegance and predictive power of physics at work to explain the incredible diversity and beauty of life, and that’s how I ended up in biophysics,” he says. Now a professor of molecular and cell biology at the University of California, Berkeley, Hurley was elected to the National Academy of Sciences in 2020. During his career, he has used precision tools, such as cryoelectron microscopy (cryo-EM), X-ray crystallography, and mass spectrometry to study the structure of proteins and membranes and the interplay between them. His work has answered fundamental questions about how these interactions influence the shape of cells and organelles and how pathogens can coopt these interactions to infect cells. In his Inaugural Article, Hurley describes the structure of an immune evasion protein of SARS-CoV-2, the virus responsible for the COVID-19 pandemic (1). After his undergraduate physics degree, Hurley studied high-energy physics at Fermilab, where he worked on a muon …

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family.

Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

A beginner’s guide to macromolecular crystallization

Obtaining diffraction-quality crystals is currently the rate-limiting step in macromolecular X-ray crystallography of proteins, DNA, RNA or their complexes, in the vast majority of cases. Since each sample has different and specific characteristics – which is the reason for wanting to study every single one of them in the first place – crystallization conditions cannot be predicted. Hence, researchers must enable crystal nucleation and growth through experimentation and screening. The size, shape and surface of the sample or complexes of interest are often altered through genetic and biochemical manipulation to facilitate crystallization, based on bioinformatics analyses and trial and error. Pure samples are trialled against a very broad range of crystallization conditions. The currently predominant method to achieve crystallization is sitting drop vapour diffusion with nanolitre-class robotic liquid handlers. Once initial screening yields crystals, further optimization experiments are usually required to obtain larger and diffraction-quality crystals.

A fully automated crystallization apparatus for small protein quantities.

In 2003, a fully automated protein crystallization and monitoring system (PXS) was developed to support the structural genomics projects that were initiated in the early 2000s. In PXS, crystallization plates were automatically set up using the vapor-diffusion method, transferred to incubators and automatically observed according to a pre-set schedule. The captured images of each crystallization drop could be monitored through the internet using a web browser. While the screening throughput of PXS was very high, the demands of users have gradually changed over the ensuing years. To study difficult proteins, it has become important to screen crystallization conditions using small amounts of proteins. Moreover, membrane proteins have become one of the main targets for X-ray crystallography. Therefore, to meet the evolving demands of users, PXS was upgraded to PXS2. In PXS2, the minimum volume of the dispenser is reduced to 0.1 µl to minimize the amount of sample, and the resolution of the captured images is increased to five million pixels in order to observe small crystallization drops in detail. In addition to the 20°C incubators, a 4°C incubator was installed in PXS2 because crystallization results may vary with temperature. To support membrane-protein crystallization, PXS2 includes a procedure for the bicelle method. In addition, the system supports a lipidic cubic phase (LCP) method that uses a film sandwich plate and that was specifically designed for PXS2. These improvements expand the applicability of PXS2, reducing the bottleneck of X-ray protein crystallography.

The Bicyclic Form of galacto-Noeurostegine Is a Potent Inhibitor of β-Galactocerebrosidase.

Competitive inhibitors of galactocerebrosidase (GALC) could be candidates for pharmacological chaperone therapy of patients with Krabbe disease. The known and selective nortropane-type iminosugar galacto-noeurostegine has been found to competitively inhibit GALC with K i = 7 μM at pH 4.6, which is 330-fold more potent than the analogous deoxynoeurostegine. It was shown through X-ray protein crystallography that galacto-noeurostegine binds to the active site of GALC in its bicyclic form.

Protein Structure Analysis and Validation with X-Ray Crystallography.

X-ray crystallography is the main technique for the determination of protein structures. About 85% of all protein structures known to date have been elucidated using X-ray crystallography. Knowledge of the three-dimensional structure of proteins can be used in various applications in biotechnology, biomedicine, drug design , and basic research and as a validation tool for protein modifications and ligand binding. Moreover, the requirement for pure, homogeneous, and stable protein solutions in crystallizations makes X-ray crystallography beneficial in other fields of protein research as well. Here, we describe the technique of X-ray protein crystallography and the steps involved for a successful three-dimensional crystal structure determination.

Post-Catalytic Complexes with Emtricitabine or Stavudine and HIV-1 Reverse Transcriptase Reveal New Mechanistic Insights for Nucleotide Incorporation and Drug Resistance.

Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a “snapshot” for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.

A new soaking procedure for X-ray crystallographic structural determination of protein–peptide complexes

Interactions between a protein and a peptide motif of its protein partner are prevalent in nature. Often, a protein also has multiple interaction partners. X-ray protein crystallography is commonly used to examine these interactions in terms of bond distances and angles as well as to describe hotspots within protein complexes. However, the crystallization process presents a significant bottleneck in structure determination since it often requires notably time-consuming screening procedures, which involve testing a broad range of crystallization conditions via a trial-and-error approach. This difficulty is also increased as each protein-peptide complex does not necessarily crystallize under the same conditions. Here, a new co-crystallization/peptide-soaking method is presented which circumvents the need to return to the initial lengthy crystal screening and optimization processes for each consequent new complex. The 14-3-3σ protein, which has multiple interacting partners with specific peptidic motifs, was used as a case study. It was found that co-crystals of 14-3-3σ and a low-affinity peptide from one of its partners, c-Jun, could easily be soaked with another interacting peptide to quickly and easily generate new structures at high resolution. Not only does this significantly reduce the production time, but new 14-3-3-peptide structures that were previously not accessible with the 14-3-3σ isoform, despite screening hundreds of other different conditions, were now also able to be resolved. The findings achieved in this study may be considered as a supporting and practical guide to potentially enable the acceleration of the crystallization process of any protein-peptide system.

Novel potent and selective pyrazolylpyrimidine-based SYK inhibitors

Hybridisation of amino-pyrimidine based SYK inhibitors (e.g. 1a) with previously reported diamine-based SYK inhibitors (e.g. TAK-659) led to the identification and optimisation of a novel pyrimidine-based series of potent and selective SYK inhibitors, where the original aminomethylene group was replaced by a 3,4-diaminotetrahydropyran group. The initial compound 5 achieved excellent SYK potency. However, it suffered from poor permeability and modest kinase selectivity. Further modifications of the 3,4-diaminotetrahydropyran group were identified and the interactions of those groups with Asp512 were characterised by protein X-ray crystallography. Further optimisation of this series saw mixed results where permeability and kinase selectivity were increased and oral bioavailability was achieved in the series, but at the expense of potent hERG inhibition.

Abstract 1792: MAPK1 p.D321N mutation confers sensitive to erlotinib in head and neck squamous cell carcinoma (HNSCC)

Abstract The annual incidence of head and neck squamous cell carcinoma (HNSCC) is ~0.88 million worldwide (IARC, WHO 2018). Despite great success of precision medicine in major cancers such as lung cancer and breast cancers, there are limited precision therapies for HNSCC. We previously reported an HNSCC patient whose tumor harbored a somatic MAPK1 p.E322K mutation demonstrating exceptional clinical response to erlotinib, an EGFR inhibitor. Subsequent laboratory investigation confirmed the ability of MAPK1 p.E322K to hyperactivate EGFR, thus causing erlotinib sensitivity in HNSCC models, both in vitro and in vivo. Here, we sequenced 105 tumors from Hong Kong (HK) HNSCC patients by next-generation sequencing (NGS) on MAPK1 gene and we found a 5.71% rate (6/105 tumors) of MAPK1 somatic mutation in HK-HNSCC. This rate appears to be relatively higher than the 1.75% (9/521 tumors) rate in the US-TCGA HNSCC cohort (P=0.0288). Apart from the MAPK1 p.E322K mutation found in the US-TCGA HNSCC cohort, we identified novel MAPK1 p.R135K and p.D321N mutations in our HK cohort which were absent in the US TCGA HNSCC. Pan-cancer mapping of MAPK1 somatic mutations revealed that MAPK1 p.R135K and p.D321N mutations are also recurrent hotspot mutation in TCGA pan-cancers (32 cancer types). Strikingly, based on the X-ray crystallography of the human MAPK1 protein, R135 and D321 amino acid residues both reside on the same kinase interaction motif (KIM) domain as E322 (all are within a 13Å distance in 3D). Thus, we further investigated if MAPK1 p.R135K and p.D321N mutations might function similarly as MAPK1 p.E322K mutant in terms of their abilities to drive HNSCC growth, and erlotinib sensitivity in HNSCC models. We employed the same FaDu retroviral infection method to study these mutations as we did previously, and we found that MAPK1 p.D321N (P<0.0001) was a driver for HNSCC proliferation, while MAPK1 p.R135K was not (all vs. MAPK1-wildtype). Subsequent in vivo erlotinib treatment experiments with xenografts bearing these mutants and MAPK1-wildtype showed that MAPK1 p.D321N tumors demonstrated heightened sensitivity to erlotinib with marked reduction of tumor size, tumor cell positivity, as well as membranous pEGFR expression when compared to vehicle treatment (P=0.0037). Whereas MAPK1 p.R135K tumors only showed a trend for erlotinib sensitivity (P=0.0646), with reduction in membranous p-EGFR expression by erlotinib in the tumors. Thus, MAPK1 p.D321N mutation confers erlotinib-sensitivity in HNSCC system. Further clinical investigation is warranted to further explore the relationship between various MAPK1 mutations and erlotinib sensitivity in HNSCC patients. Citation Format: Hoi Lam Ngan, Peony Hiu Yan Poon, Yu-Xiong Su, Jason Yik Kuen Chan, Kwok-Wai Lo, Chun Kit Yeung, Yuchen Liu, Eileen Wong, Hui Li, Chin Wang Lau, Wenying Piao, Vivian Wai Yan Lui. MAPK1 p.D321N mutation confers sensitive to erlotinib in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1792.

A chemical interpretation of protein electron density maps in the worldwide protein data bank.

High-quality three-dimensional structural data is of great value for the functional interpretation of biomacromolecules, especially proteins; however, structural quality varies greatly across the entries in the worldwide Protein Data Bank (wwPDB). Since 2008, the wwPDB has required the inclusion of structure factors with the deposition of x-ray crystallographic structures to support the independent evaluation of structures with respect to the underlying experimental data used to derive those structures. However, interpreting the discrepancies between the structural model and its underlying electron density data is difficult, since derived sigma-scaled electron density maps use arbitrary electron density units which are inconsistent between maps from different wwPDB entries. Therefore, we have developed a method that converts electron density values from sigma-scaled electron density maps into units of electrons. With this conversion, we have developed new methods that can evaluate specific regions of an x-ray crystallographic structure with respect to a physicochemical interpretation of its corresponding electron density map. We have systematically compared all deposited x-ray crystallographic protein models in the wwPDB with their underlying electron density maps, if available, and characterized the electron density in terms of expected numbers of electrons based on the structural model. The methods generated coherent evaluation metrics throughout all PDB entries with associated electron density data, which are consistent with visualization software that would normally be used for manual quality assessment. To our knowledge, this is the first attempt to derive units of electrons directly from electron density maps without the aid of the underlying structure factors. These new metrics are biochemically-informative and can be extremely useful for filtering out low-quality structural regions from inclusion into systematic analyses that span large numbers of PDB entries. Furthermore, these new metrics will improve the ability of non-crystallographers to evaluate regions of interest within PDB entries, since only the PDB structure and the associated electron density maps are needed. These new methods are available as a well-documented Python package on GitHub and the Python Package Index under a modified Clear BSD open source license.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a “shield” in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

Crystallographic Structure Determination of Bacteriophage Endolysins.

Bacteriophages produce endolysins that target and cleave the hosts peptidoglycan to release their progeny at the end of the infection cycle. These proteins can be used for the eradication of pathogenic bacteria, but also for their detection. Endolysins may contain a single catalytic domain or several domains, including a cell wall binding domain. To understand their function in detail and design mutated or chimeric molecules with novel properties, knowledge of their structures and detailed mechanisms is necessary. X-ray protein crystallography is an excellent method to obtain high-resolution structures of biological macromolecules, and here we describe the method and the folds of known endolysin domains.

Self-Immolation of a Bacterial Dehydratase Enzyme by its Epoxide Product.

Disabling the bacterial capacity to cause infection is an innovative approach that has attracted significant attention to fight against superbugs. A relevant target for anti-virulence drug discovery is the type I dehydroquinase (DHQ1) enzyme. It was shown that the 2-hydroxyethylammonium derivative 3 has in vitro activity since it causes the covalent modification of the catalytic lysine residue of DHQ1. As this compound does not bear reactive electrophilic centers, how the chemical modification occurs is intriguing. We report here an integrated approach, which involves biochemical studies, X-ray crystallography and computational studies on the reaction path using combined quantum mechanics/molecular mechanics Umbrella Sampling Molecular Dynamics, that evidences that DHQ1 catalyzes its self-immolation by transforming the unreactive 2-hydroxyethylammonium group in 3 into an epoxide that triggers the lysine covalent modification. This finding might open opportunities for the design of lysine-targeted irreversible inhibitors bearing a 2-hydroxyethylammonium moiety as an epoxide proform, which to our knowledge has not been reported previously.

Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography.

Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.

Protein X-ray Crystallography and Drug Discovery.

With the advent of structural biology in the drug discovery process, medicinal chemists gained the opportunity to use detailed structural information in order to progress screening hits into leads or drug candidates. X-ray crystallography has proven to be an invaluable tool in this respect, as it is able to provide exquisitely comprehensive structural information about the interaction of a ligand with a pharmacological target. As fragment-based drug discovery emerged in the recent years, X-ray crystallography has also become a powerful screening technology, able to provide structural information on complexes involving low-molecular weight compounds, despite weak binding affinities. Given the low numbers of compounds needed in a fragment library, compared to the hundreds of thousand usually present in drug-like compound libraries, it now becomes feasible to screen a whole fragment library using X-ray crystallography, providing a wealth of structural details that will fuel the fragment to drug process. Here, we review theoretical and practical aspects as well as the pros and cons of using X-ray crystallography in the drug discovery process.

Probing the role of the conserved residue Glu166 in a class A β-lactamase using neutron and X-ray protein crystallography.

The amino-acid sequence of the Toho-1 β-lactamase contains several conserved residues in the active site, including Ser70, Lys73, Ser130 and Glu166, some of which coordinate a catalytic water molecule. This catalytic water molecule is essential in the acylation and deacylation parts of the reaction mechanism through which Toho-1 inactivates specific antibiotics and provides resistance to its expressing bacterial strains. To investigate the function of Glu166 in the acylation part of the catalytic mechanism, neutron and X-ray crystallographic studies were performed on a Glu166Gln mutant. The structure of this class A β-lactamase mutant provides several insights into its previously reported reduced drug-binding kinetic rates. A joint refinement of both X-ray and neutron diffraction data was used to study the effects of the Glu166Gln mutation on the active site of Toho-1. This structure reveals that while the Glu166Gln mutation has a somewhat limited impact on the positions of the conserved amino acids within the active site, it displaces the catalytic water molecule from the active site. These subtle changes offer a structural explanation for the previously observed decreases in the binding of non-β-lactam inhibitors such as the recently developed diazobicyclooctane inhibitor avibactam.

Protein Footprinting and X-ray Crystallography Reveal the Interaction of PD-L1 and a Macrocyclic Peptide.

Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, small-molecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small-molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP), and corroborated the findings with our X-ray structure of the PD-L1/macrocycle complex. Although all three approaches show that the macrocycle binds directly to PD-L1 over the regions of residues 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting in probing protein/ligand inhibitory interactions in cancer immunotherapy.