Project description:The identification of weak-affinity ligands targeting membrane proteins is of great interest in Fragment-Based Drug Design (FBDD). Recently, miniaturized weak affinity chromatography (WAC) has been proposed as a valuable tool to study interactions between small ligands and wild-type membrane proteins embedded in so-called nanodisc biomimetic membranes immobilized on GMA-co-EDMA monoliths in situ-synthesized in capillary columns (less than one microliter in volume). In this proof-of-concept study, the achievable affinity range was limited to medium affinity (low micromolar range). The present work investigates different strategies to extend the affinity range towards low affinities, either by increasing the density of membrane proteins on the chromatographic support or by reducing non-specific interactions with the monolith. The combination of the use of a new and more hydrophilic monolithic support (poly(DHPMA-co-MBA)) and a multilayer nanodisc grafting process (up to three layers) allows a significant increase in the membrane protein density by a more than three-fold factor (up to 5.4 pmol cm-1). Such an increase in protein density associated with reduced non-specific interactions makes it possible to extend the range of detectable affinity, as demonstrated by the identification and characterization of affinities of very low-affinity ligands (Kd values of several hundred micromolar) for the adenosine receptor AA2AR used as a model protein, which was not possible before. The affinity was confirmed by competition experiments.
Project description:Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.
Project description:Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3-12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F'- and G'-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4-membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.
Project description:The C1 domain, which represents the recognition motif on protein kinase C for the lipophilic second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters, has emerged as a promising therapeutic target for cancer and other indications. Potential target selectivity is markedly enhanced both because binding reflects ternary complex formation between the ligand, C1 domain, and phospholipid, and because binding drives membrane insertion of the C1 domain, permitting aspects of the C1 domain surface outside the binding site, per se, to influence binding energetics. Here, focusing on charged residues identified in atypical C1 domains which contribute to their loss of ligand binding activity, we showed that increasing charge along the rim of the binding cleft of the protein kinase C δ C1 b domain raises the requirement for anionic phospholipids. Correspondingly, it shifts the selectivity of C1 domain translocation to the plasma membrane, which is more negatively charged than internal membranes. This change in localization is most pronounced in the case of more hydrophilic ligands, which provide weaker membrane stabilization than do the more hydrophobic ligands and thus contributes an element to the structure-activity relations for C1 domain ligands. Coexpressing pairs of C1-containing constructs with differing charges each expressing a distinct fluorescent tag provided a powerful tool to demonstrate the effect of increasing charge in the C1 domain.
Project description:Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, Yersinia pestis type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).
Project description:Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.
Project description:Ligands that have an affinity for protein targets can be screened very effectively by exploiting favorable properties of long-lived states (LLS) in NMR spectroscopy. In this work, we describe the use of LLS for competitive binding experiments to measure accurate dissociation constants of fragments that bind weakly to the ATP binding site of the N-terminal ATPase domain of heat shock protein 90 (Hsp90), a therapeutic target for cancer treatment. The LLS approach allows one to characterize ligands with an exceptionally wide range of affinities, since it can be used for ligand concentrations [L] that are several orders of magnitude smaller than the dissociation constants K(D). This property makes the LLS method particularly attractive for the initial steps of fragment-based drug screening, where small molecular fragments that bind weakly to a target protein must be identified, which is a difficult task for many other biophysical methods.
Project description:The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel family (pLGIC), displays remarkable variations in the affinity and efficacy of the full agonist glycine and the partial agonist taurine depending on the cell system used. Despite detailed insights in the GlyR three-dimensional structure and activation mechanism, little is known about conformational rearrangements induced by these agonists. Here, we characterized the conformational states of the α1 GlyR upon binding of glycine and taurine by microscale thermophoresis expressed in HEK293 cells and Xenopus oocytes after solubilization in amphipathic styrene-maleic acid copolymer nanodiscs. Our results show that glycine and taurine induce different conformational transitions of the GlyR upon ligand binding. In contrast, the variability of agonist affinity is not mediated by an altered conformational change. Thus, our data shed light on specific agonist induced conformational features and mechanisms of pLGIC upon ligand binding determining receptor activation in native environments.
Project description:From the effects of N-acetyl-d-glucosamine and d-glucose on the elution of the A protein of human lactose synthetase from a column of Sepharose-alpha-lactalbumin, values of 200m(-1) and 0.57m(-1) are deduced for the association constants describing the interaction between the enzyme and the respective monosaccharides.
Project description:Cytochrome P4503A4 (CYP3A4) is a peripheral membrane protein that plays a major role in enzymatic detoxification of many drugs and toxins. CYP3A4 has an integral membrane N-terminal helix and a localized patch comprised of the G' and F' helix regions that are embedded in the membrane, but the effects of membrane composition on CYP3A4 function are unknown. Here, circular dichroism and differential scanning calorimetry were used to compare the stability of CYP3A4 in lipid bilayer nanodiscs with varying ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These lipids differ in the acyl-chain length and their degree of unsaturation. The thermal denaturation of CYP3A4 in nanodiscs occurs in a temperature range distinct from that of the nanodisc denaturation so it can be monitored calorimetrically. Melting temperatures (Tm), heat capacities (ΔCp), and calorimetric enthalpies (ΔHcal) for denaturation of CYP3A4 each increased with an increasing fraction of DMPC, with a maximum at 50% DMPC, before decreasing at 75% DMPC. Addition of the inhibitor ketoconazole results in increased thermal stability, and larger ΔCp and ΔHcal values, with different sensitivities to lipid composition. Effects of lipid composition on ligand binding dynamics were also studied. Equilibrium binding affinities of both ketoconazole (KTZ) and testosterone (TST) were minimally affected by lipid composition. However, stopped-flow analyses indicate that the rates of KTZ binding reach a maximum in membranes containing 50% DMPC, whereas the rate of TST binding decreases continuously with an increasing DMPC concentration. These results indicate that CYP3A4 is highly sensitive to the acyl-chain composition of the lipids and fluidity of the membrane in which it is embedded.