Combining MicroED and native mass spectrometry for structural discovery of enzyme–small molecule complexes

β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high risk factor in Parkinson's disease. The implementation of a small molecule modulator is a strategy to restore proper folding and lysosome delivery of degradation-prone mutant GCase. Here, we present a potent quinazoline modulator, JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase abundance in patient-derived fibroblast cells. We then developed a covalent modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro mechanistic studies. By using native top-down mass spectrometry, we located two potentially covalently modified lysines. We obtained the first crystal structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator covalently bound, and were able to identify the exact lysine residue modified (Lys346) and reveal an allosteric binding site. GCase dimerization was induced by our modulator binding, which was observed by native mass spectrometry, its crystal structure, and size exclusion chromatography with a multiangle light scattering detector. Finally, the dimer form was confirmed by negative staining transmission electron microscopy studies. Our newly discovered allosteric site and observed GCase dimerization provide a new mechanistic insight into GCase and its noniminosugar modulators and facilitate the rational design of novel GCase modulators for Gaucher's disease and Parkinson's disease.

Molecular glues stabilize interactions between E3 ligases and novel substrates to promote substrate degradation, thereby facilitating the inhibition of traditionally "undruggable" protein targets. However, most known molecular glues have been discovered fortuitously or are based on well-established chemical scaffolds. Efficient approaches for discovering and characterizing the effects of molecular glues on protein interactions are required to accelerate the discovery of novel agents. Here, we demonstrate that native mass spectrometry and mass photometry can provide unique insights into the physical mechanism of molecular glues, revealing previously unknown effects of such small molecules on the oligomeric organization of E3 ligases. When compared to well-established solution phase assays, native mass spectrometry provides accurate quantitative descriptions of molecular glue potency and efficacy while also enabling the binding specificity of E3 ligases to be determined in a single, rapid measurement. Such mechanistic insights should accelerate the rational development of molecular glues to afford powerful therapeutic agents.

Integral membrane proteins are embedded in biological membranes where various lipids modulate their structure and function. There exists a critical need to elucidate how these lipids participate in the physiological and pathological processes associated with the membrane protein dysfunction. Native mass spectrometry (MS), combined with ion mobility spectrometry (IM), is emerging as a powerful tool to probe membrane protein complexes and their interactions with ligands, lipids, and other small molecules. Unlike other biophysical approaches, native IM-MS can resolve individual ligand/lipid binding events. We have developed a novel method using native MS, coupled with a temperature-control apparatus, to determine the thermodynamic parameters of individual ligand or lipid binding events to proteins. This approach has been validated using several soluble protein-ligand systems wherein MS results are compared with those acquired from conventional biophysical techniques, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Using these principles, it is possible to elucidate the thermodynamics of individual lipid binding to integral membrane proteins. Herein, we use the ammonia channel (AmtB) from Escherichia coli as a model membrane protein. Remarkably, distinct thermodynamic signatures for AmtB binding to lipids with different headgroups and acyl chain configurations are observed. Additionally, using a mutant form of AmtB that abolishes a specific lipid binding site, distinct changes have been discovered in the thermodynamic signatures compared with the wild-type, implying that these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites. This chapter provides procedures and findings associated with temperature-controlled native MS as a novel approach to interrogate membrane proteins and their interactions with lipids and other molecules.

A new aluminosilicate zeolite, denoted EMM-37, with a 3D small pore channel system, has been synthesized using a diquaternary ammonium molecule as the structure directing agent (SDA) and metakaolin as the aluminum source. The structures of both as-made and calcined forms of EMM-37 were solved and refined using continuous rotation electron diffraction (cRED) data. cRED is a powerful method for the collection of 3D electron diffraction data from submicron- and nanosized crystals, which allows for successful solution and refinement of complex structures in symmetry as low as P1̅.

Targeting nucleic acids with small molecules has shown strong potential as a therapeutic strategy. However, before this potential can be realized, the interactions between the small molecule and the nucleic acid target must be fully characterized. Trinucleotide repeat diseases provide a good model system for studying the interactions between small molecules and target nucleic acids. The d(CTG·CAG)exp repeat that causes myotonic dystrophy type 1 and Huntington’s Disease is one such target. Herein, we used an interdisciplinary approach that combined solution-phase stability assays, native mass spectrometry, and enzymatic transcription assays to study the interactions between three unique compounds and disease-associated repeat expansion DNA. Stability assays and native mass spectrometry measurements evaluated the nature of interactions occurring between the compounds studied and DNA and determined that two of the compounds are highly prone to interacting with DNA, with one compound demonstrating some degree of specificity to repeating d(CTG) sequences. Transcription assays indicated that only one of the three compounds studied herein effectively inhibits bidirectional transcription of only repeat expansion containing DNA. The data supports that the lead compound interacts with the disease-relevant repeat DNA and shows promise as a potential therapeutic agent.

Among the drug targets being investigatedfor SARS-CoV-2, the viralmain protease (Mpro) is one of the most extensively studied.Mpro is a cysteine protease that hydrolyzes the viral polyproteinat more than 11 sites. It is highly conserved and has a unique substratepreference for glutamine in the P1 position. Therefore, Mpro inhibitors are expected to have broad-spectrum antiviral activityand a high selectivity index. Structurally diverse compounds havebeen reported as Mpro inhibitors. In this study, we investigatedthe mechanism of action of six previously reported Mpro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin,and PX-12, using a consortium of techniques including FRET-based enzymaticassay, thermal shift assay, native mass spectrometry, cellular antiviralassays, and molecular dynamics simulations. Collectively, the resultsshowed that the inhibition of Mpro by these six compoundsis nonspecific and that the inhibition is abolished or greatly reducedwith the addition of reducing reagent 1,4-dithiothreitol (DTT). WithoutDTT, these six compounds inhibit not only Mpro but alsoa panel of viral cysteine proteases including SARS-CoV-2 papain-likeprotease and 2Apro and 3Cpro from enterovirusA71 (EV-A71) and EV-D68. However, none of the compounds inhibits theviral replication of EV-A71 or EV-D68, suggesting that the enzymaticinhibition potency IC50 values obtained in the absenceof DTT cannot be used to faithfully predict their cellular antiviralactivity. Overall, we provide compelling evidence suggesting thatthese six compounds are nonspecific SARS-CoV-2 Mpro inhibitorsand urge the scientific community to be stringent with hit validation.

Cells maintain protein homeostasis by adding a small protein, ubiquitin, to regulate a variety of cellular processes, dictating protein activity, localisation or degradation. The addition of ubiquitin, known as ubiquitylation, is a reversible process making it a versatile post‐translational modification aptly suited for cell signalling. Removal of ubiquitin is catalysed by deubiquitylating enzymes, commonly referred to as DUBs. BRCC36 isopeptidase complex (BRISC) is a multi‐protein DUB complex which hydrolyses lysine‐63‐linked ubiquitin chains on Type I interferon receptors (IFNAR1/2), thus regulating interferon‐dependent signalling. Therefore, BRISC‐mediated inflammatory signalling amplification is a promising target for autoimmune disease drug development. We performed a high‐throughput screen to identify small molecules which inhibit BRISC enzymatic activity. Employing an integrative structural biology approach (cryo‐electron microscopy, native mass spectrometry, hydrogen‐deuterium exchange mass spectrometry), complemented with biochemical assays, we have uncovered new enzyme conformations, revealing a remarkable mode of action for BRISC inhibitors. Exploring these key mechanisms will expand current knowledge of inflammatory signalling pathways and establish the use of DUB inhibitors as therapeutics to combat autoimmune disease and hyperactive cytokine signalling.

Considering that aprotic solvents are often used as cosolvents in investigating the interactions between small molecules and proteins, we assessed the effects of five aprotic solvents represented by dimethylformamide (DMF) on the structure stabilities of metal-free SOD1 (apo-SOD1) by native electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS). These aprotic solvents include DMF, 1,3-dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide (DMSO), acetonitrile (ACN), and tetrahydrofuran (THF). Results indicated that DMI, DMSO, and DMF at low percentage concentration could reduce the average charge and the dimer dissociation of apo-SOD1. By contrast, ACN and THF at low concentration have no similar effect. DMF was selected as a representative solvent to further investigate the detailed effects on the structure stability of apo-SOD1 by using collision-induced dissociation and unfolding. The results reveal that the addition of minimal DMF to an aqueous protein solution can protect against the unfolding and dissociation of dimer, even under destabilizing conditions (such as low pH or high cone voltage). When the different percentage concentrations of DMF were added, the average collision cross section of apo-SOD1 showed that apo-SOD1 became compacted when the DMF concentration increased from 0% to 1% and eventually started extending when increased from 1% to 20%. The results indicated that DMF has similar effects to DMSO in native mass spectrometry (MS) and it can also be used as a cosolvent besides DMSO in investigating the stabilities of proteins and the interactions between small molecules and proteins.

Small molecules inhibit the interaction of Nrf2 and the Keap1 Kelch domain through a non-covalent mechanism

Electron diffraction emerges as a powerful technique for structural analysis of small crystals, especially those that are too small for single crystal X-ray analysis or too complex for powder diffraction. Its growing popularity is driven by the strong electron-matter interaction and the opportunity for single-crystal data collection from nanosized crystals. However, this strong interaction often comes with the caveat of possible damage to the sample by the electron beam, a drawback that can affect the crystal structure and compromise data quality. This review delves into the details of the effects of beam damage on electron diffraction data, particularly focusing on the fading of Bragg reflections that are known to be the most sensitive damage indicator. By compiling the observations from quantitative measurements across the available reports, a treatise is provided on the effects of electron beam damage on electron diffraction data. Comparison of various mitigation strategies is also provided which sets guidelines for optimization of data collection strategies for efficient exposure of beam-sensitive compounds. We hope that this review will provide valuable insights forthe growing research community that resortsto electron diffraction for characterization of materials, sometimes as the only applicable method to determine the structure of very small crystals.

These past few years, many new structures have been solved using electron diffraction methods: zone axis precession electron diffraction (PED) and tomography in reciprocal space. Both methods enable to reduce importantly the multiple scattering, so that the reflection intensities can be used for structure determination by direct methods. The ferrite Sr25Fe30O77 belongs to a family of phases whose structures consist of an intergrowth of m perovskite layers with complex rocksalt type layers [1,2]. The compound of interest is the member m = 4 of this family and its structure has been solved by combining both electron diffraction methods cited above. This oxide crystallizes in an orthorhombic system with the sub-cell parameters a ≍ b ≍ 5.4 Å and c ≍ 42 Å in a F type lattice. The structure exhibits modulation along a axis with a modulation vector q = 2/5 a. The commensurate nature of the modulation enables to describe the structure in a supercell with the cell parameters a ≍ 27 Å, b ≍ 5.4 Å and c ≍ 42 Å. PED patterns were recorded in zone axis with a Spinning Star unit using a precession angle of 20. The intensities were extracted with CRISP software and the resulting dataset was then implemented in SIR2008 for structure solution. The tomography data collection, recorded by tilting manually every 0.5 degree from -30 to +30 degrees, was inserted in a "3D Electron Diffraction Tomography" software, which reconstructs the 3D reciprocal space and extracts automatically the reflection intensities. The intensity file was then used on SIR2008 for structure determination. In order to confirm and refine the structural model, a powder X-ray diffraction pattern was performed on a laboratory diffractometer with Cu Ka1 radiation. Cell parameters were refined with the WinPlotr and FullProf Softwares using both LeBail and Rietveld methods [3]. The structural model obtained with electron diffraction data was tried and confirmed as the correct structure by the Rietveld refinements.

Molecular glues are powerful tools for the control of protein-protein interactions. Yet, the mechanisms underlying multi-component protein complex formation remain poorly understood. Native mass spectrometry (MS) detects multiple protein species simultaneously, providing an entry to elucidate these mechanisms. Here, for the first time, covalent molecular glue stabilization was kinetically investigated by combining native MS with biophysical and structural techniques. This approach elucidated the stoichiometry of a multi-component protein-ligand complex, the assembly order, and the contributions of covalent versus non-covalent binding events that govern molecular glue activity. Aldehyde-based molecular glue activity is initially regulated by cooperative non-covalent binding, followed by slow covalent ligation, further enhancing stabilization. This study provides a framework to investigate the mechanisms of covalent small molecule ligation and informs (covalent) molecular glue development.

A large number of studies have shown that the production of aberrant and deleterious copper zinc superoxide dismutase (SOD1) species is closely related to amyotrophic lateral sclerosis (ALS). Therefore, it is of great significance to screen effective inhibitors of misfolding and aggregation of SOD1 for treating ALS disease. The interaction between flavanone compounds with apo-SOD1was investigated using native electrospray ion mobility mass spectrometry (native ESI-IM-MS). Binding affinities of ligands were compared using native MS, ESI-MS/MS, collision-induced unfolding, and competitive experiments. The effect of ligands on apo-SOD1 aggregation was investigated using the fluorescence spectroscopy method. The results of MS showed that the binding affinity of liquiritin apioside was the strongest, better than the corresponding monosaccharide and aglycone, indicating that the presence and the number of glycosyl group are beneficial to enhance ligand affinity to protein. The results of fluorescence spectroscopy for inhibiting protein aggregation in vitro were consistent with the binding affinity. In addition, the results of the collision-induced unfolding indicated that liquiritin apioside can slow down the unfolding of the protein. Meanwhile, the results of competition experiment suggested that liquiritin apiosides share different binding sites with naringin and 5-fluorouridine, which are significant for the structural stability of SOD1. This study revealed that the binding of liquiritin apioside can stabilize apo-SOD1 dimer and inhibit the aggregation of apo-SOD1, and illustrated that native ESI-IM-MS is a powerful tool for providing insight into investigating the structure-activity relationship between small molecules and protein, and screening protein conformation stabilizers.

Overexpression of anti-apoptotic proteins such as BCL-xL is a hallmark of various cancers and a major driver of resistance to conventional chemotherapies. While small-molecule BCL-xL inhibitors have shown promising outcomes, their clinical use is hindered by dose-limiting toxicities, especially thrombocytopenia. Proteolysis-targeting chimeras (PROTACs) offer a promising alternative by promoting selective degradation of target proteins via the ubiquitin-proteasome system, thereby reducing off-target effects associated with small molecule inhibitors. However, rational design and optimization of PROTACs remain challenging due to the need to balance simultaneous interactions with both an E3 ubiquitin ligase and the target protein. Here we employ native mass spectrometry (MS) as a rapid, label-free platform to screen and characterize the formation and stability of ternary complexes between BCL-xL, VHL E3 ligase complex (VCB), and various targeting PROTACs. Native MS enables direct detection of binary BCL-xL●PROTAC and ternary BCL-xL●PROTAC●VCB complexes and provides semi-quantitative insights into PROTAC affinity and cooperativity with both binding partners. Furthermore, we explore the dissociation pathways of these complexes in the gas phase using collision-induced dissociation (CID) and ultraviolet photodissociation (UVPD), revealing distinct fragmentation and subunit release patterns that reflect the structural organization and gas-phase stability of the complexes. Variable-temperature ESI-MS (vT-ESI) further allows assessment of thermal stabilities of the complexes in solution. Together, our study demonstrates the power of native MS to both screen and mechanistically characterize PROTAC-induced ternary complex formation.

Invasive fungal infections (IFIs) are primarily caused by Candida spp., Cryptococcus neoformans, Aspergillus spp., Mucor spp., Sporothrix spp., and Pneumocystis spp., which attack human organs with a strong pathogenicity and exhibit drug resistance against commonly used chemical drugs. Therefore, the search for alternative drugs with high efficacy, low resistance rates, few side effects, and synergistic antifungal effects remains a major challenge. The characteristics of natural products with structural and bioactive diversity, lower drug resistance, and rich resources make them a major focus of the development of antifungal drugs. This review attempts to summarize the origin, structure, and antifungal activity of natural products and their derivatives with MIC ≤ 20 µg/mL or 100 µM, focusing on their MoA and SAR. All pertinent literature databases were searched. The search keywords were antifungal or antifungals, terpenoids, steroidal saponins, alkaloid, phenols, lignans, flavonoids, quinones, macrolide, peptide, tetramic acid glycoside, polyene, polyketide, bithiazole, natural product, and derivatives. All the related literature (covering the past 20 years, 2001-2022) was evaluated. In total, 340 natural products and 34 synthesized derivatives with antifungal activity from 301 studies were included in this review. These compounds were derived from terrestrial plants, ocean life, and microorganisms and exhibited in vitro and in vivo potent antifungal activity alone or in combination. The MoA and SARs of reported compounds were summarized whenever applicable. In this review, we attempted to review the available literature on natural antifungal products and their derivatives. Most of the studied compounds showed potent activity against Candida species, Aspergillus species, or Cryptococcus species. Some of the studied compounds also demonstrated the ability to impair the cell membrane and cell wall, inhibit hypha and biofilms, and cause mitochondrial dysfunction. Although the MoAs of these compounds are not well understood yet, they can be used as lead components for the development of new, effective, and safe antifungal agents through their novel mechanisms.