High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [ß1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using ß1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαißγ and ß-arrestin-1 and showed that carvedilol induces an increase in coupling of ß-arrestin-1 and Gαißγ to ß1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry.

Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

Identification of Chemokine Ligands by Biochemical Fragmentation and Simulated Peptide Evolution.

Short linear peptides can overcome certain limitations of small molecules for targeting protein-protein interactions (PPIs). Herein, the interaction between the human chemokine CCL19 with chemokine receptor CCR7 was investigated to obtain receptor-derived CCL19-binding peptides. After identifying a linear binding site of CCR7, five hexapeptides binding to CCL19 in the low micromolar to nanomolar range were designed, guided by pharmacophore and lipophilicity screening of computationally generated peptide libraries. The results corroborate the applicability of the computational approach and the chosen selection criteria to obtain short linear peptides mimicking a protein-protein interaction site.

Binding Specificities of Nanobody•Membrane Protein Complexes Obtained from Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry.

The application of nanobodies as binding partners for structure stabilization in protein X-ray crystallography is taking an increasingly important role in structural biology. However, the addition of nanobodies to the crystallization matrices might complicate the optimization of the crystallization process, which is why analytical techniques to screen and characterize suitable nanobodies are useful. Here, we show how chemical cross-linking combined with high-mass matrix-assisted laser/desorption ionization mass spectrometry can be employed as a fast screening technique to determine binding specificities of intact nanobody•membrane protein complexes. Titration series were performed to rank the binding affinity of the interacting nanobodies. To validate the mass spectrometry data, microscale thermophoresis was used, which showed binding affinities of the stronger binding nanobodies, in the low µM range. In addition, mass spectrometry provides access to the stoichiometry of the complexes formed, which enables the definition of conditions under which homogeneous complex states are present in solution. Conformational changes of the membrane protein were investigated and competitive binding experiments were used to delimit the interaction sites of the nanobodies, which is in agreement with crystal structures obtained. The results show the diversity of specifically binding nanobodies in terms of binding affinity, stoichiometry, and binding site, which illustrates the need for an analytical screening approach.

De Novo Fragment Design for Drug Discovery and Chemical Biology.

Automated molecular de novo design led to the discovery of an innovative inhibitor of death-associated protein kinase 3 (DAPK3). An unprecedented crystal structure of the inactive DAPK3 homodimer shows the fragment-like hit bound to the ATP pocket. Target prediction software based on machine learning models correctly identified additional macromolecular targets of the computationally designed compound and the structurally related marketed drug azosemide. The study validates computational de novo design as a prime method for generating chemical probes and starting points for drug discovery.

Leveraging molecular structure and bioactivity with chemical language models for de novo drug design.

Generative chemical language models (CLMs) can be used for de novo molecular structure generation by learning from a textual representation of molecules. Here, we show that hybrid CLMs can additionally leverage the bioactivity information available for the training compounds. To computationally design ligands of phosphoinositide 3-kinase gamma (PI3Kγ), a collection of virtual molecules was created with a generative CLM. This virtual compound library was refined using a CLM-based classifier for bioactivity prediction. This second hybrid CLM was pretrained with patented molecular structures and fine-tuned with known PI3Kγ ligands. Several of the computer-generated molecular designs were commercially available, enabling fast prescreening and preliminary experimental validation. A new PI3Kγ ligand with sub-micromolar activity was identified, highlighting the method's scaffold-hopping potential. Chemical synthesis and biochemical testing of two of the top-ranked de novo designed molecules and their derivatives corroborated the model's ability to generate PI3Kγ ligands with medium to low nanomolar activity for hit-to-lead expansion. The most potent compounds led to pronounced inhibition of PI3K-dependent Akt phosphorylation in a medulloblastoma cell model, demonstrating efficacy of PI3Kγ ligands in PI3K/Akt pathway repression in human tumor cells. The results positively advocate hybrid CLMs for virtual compound screening and activity-focused molecular design.

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth.

PURPOSE: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. METHODS: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. RESULTS: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. CONCLUSIONS: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.

Structural insights into the interaction of botulinum neurotoxin a with its neuronal receptor SV2C.

A dual-receptor interaction with a polysialoganglioside and synaptic vesicle glycoprotein 2 (SV2) is required for botulinum neurotoxin A (BoNT) toxicity. Here, we review what is currently known about the BoNT/A-SV2 interaction based on structural studies. Currently, five crystal structures of the receptor-binding domain (Hc) of BoNT subtypes A1 and A2 complexed to the large luminal domain (LD4) of SV2C have been determined. On the basis of the available structures, we will discuss the importance of protein-protein and protein-carbohydrate interactions for BoNT/A toxicity as well as the high plasticity of BoNT/A for receptor recognition by tolerating a variety of side-chain interactions at the interface. A plausible explanation how receptor-binding specificity of BoNT/A may be achieved without an extensive and conserved side chain-side chain interaction network will be provided.

Interaction analysis of glycoengineered antibodies with CD16a: a native mass spectrometry approach.

Minor changes in the quality of biologically manufactured monoclonal antibodies (mAbs) can affect their bioactivity and efficacy. One of the most important variations concerns the N-glycosylation pattern, which directly affects an anti-tumor mechanism called antibody-dependent cell-meditated cytotoxicity (ADCC). Thus, careful engineering of mAbs is expected to enhance both protein-receptor binding and ADCC. The specific aim of this study is to evaluate the influence of terminal carbohydrates within the Fc region on the interaction with the FcγRIIIa/CD16a receptor in native and label-free conditions. The single mAb molecule comprises variants with minimal and maximal galactosylation, as well as α2,3 and α2,6-sialic acid isomers. Here, we apply native electrospray ionization mass spectrometry to determine the solution-phase antibody-receptor equilibria and by using temperature-controlled nanoelectrospray, a thermal stability of the complex is examined. Based on these, we prove that the galactosylation of a fucosylated Fc region increases the binding to CD16a 1.5-fold when compared with the non-galactosylated variant. The α2,6-sialylation has no significant effect on the binding, whereas the α2,3-sialylation decreases it 1.72-fold. In line with expectation, the galactoslylated and α2,6-sialylated mAb:CD16a complex exhibit higher thermal stability when measured in the temperature gradient from 20 to 50°C. The similar binding pattern is observed based on surface plasmon resonance analysis and immunofluorescence staining using natural killer cells. The results of our study provide new insight into N-glycosylation-based interaction of the mAb:CD16a complex.

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding.

Helicobacter pylori (H. pylori) secretes the chaperone and serine protease high temperature requirement A (HtrA) that cleaves gastric epithelial cell surface proteins to disrupt the epithelial integrity and barrier function. First inhibitory lead structures have demonstrated the essential role of HtrA in H. pylori physiology and pathogenesis. Comprehensive drug discovery techniques allowing high-throughput screening are now required to develop effective compounds. Here, we designed a novel fluorescence resonance energy transfer (FRET) peptide derived from a gel-based label-free proteomic approach (direct in-gel profiling of protease specificity) as a valuable substrate for H. pylori HtrA. Since serine proteases are often sensitive to metal ions, we investigated the influence of different divalent ions on the activity of HtrA. We identified Zn++ and Cu++ ions as inhibitors of H. pylori HtrA activity, as monitored by in vitro cleavage experiments using casein or E-cadherin as substrates and in the FRET peptide assay. Putative binding sites for Zn++ and Cu++ were then analyzed in thermal shift and microscale thermophoresis assays. The findings of this study will contribute to the development of novel metal ion-dependent protease inhibitors, which might help to fight bacterial infections.

When barriers ignore the "rule-of-five".

Why are a few drugs with properties beyond the rule of 5 (bRo5) absorbed across the intestinal mucosa while most other bRo5 compounds are not? Are such exceptional bRo5 compounds exclusively taken up by carrier-mediated transport or are they able to permeate the lipid bilayer (passive lipoidal diffusion)? Our experimental data with liposomes indicate that tetracycline, which violates one rule of the Ro5, and rifampicin, violating three of the rules, significantly permeate a phospholipid bilayer with kinetics similar to labetalol and metoprolol, respectively. Published data from experimental work and molecular dynamics simulations suggest that the formation of intramolecular H-bonds and the possibility to adopt an elongated shape besides the presence of a significant fraction of net neutral species facilitate lipid bilayer permeation. As an alternative to lipid bilayer permeation, carrier proteins can be targeted to improve absorption, with the potential drawbacks of drug-drug interactions and non-linear pharmacokinetics.