A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions

Biophysical label-free assays such as those based on SPR are essential tools in generating high-quality data on affinity, kinetic, mechanistic and thermodynamic aspects of interactions between target proteins and potential drug candidates. Here we show examples of the integration of SPR with bioinformatic approaches and mutation studies in the early drug discovery process. We call this combination 'structure-based biophysical analysis'. Binding sites are identified on target proteins using information that is either extracted from three-dimensional structural analysis (X-ray crystallography or NMR), or derived from a pharmacore model based on known binders. The binding site information is used for in silico screening of a large substance library (e.g. available chemical directory), providing virtual hits. The three-dimensional structure is also used for the design of mutants where the binding site has been impaired. The wild-type target and the impaired mutant are then immobilized on different spots of the sensor chip and the interactions of compounds with the wild-type and mutant are compared in order to identify selective binders for the binding site of the target protein. This method can be used as a cost-effective alternative to high-throughput screening methods in cases when detailed binding site information is available. Here, we present three examples of how this technique can be applied to provide invaluable data during different phases of the drug discovery process.     

Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors

The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.     

Cisplatin Affects the Conformation of Apo Form,not Holo Form,of BRCA1 RING Finger Domain and Confers Thermal Stability

The breast cancer suppressor protein 1 (BRCA1) has been shown to participate in genomic integrity maintenance. Preclinical and clinical studies have recently revealed that the inactivation of BRCA1 in cancer cells leads to chemosensitivity. Approaching the BRCA1 RING protein as a potentially molecular target for a platinum‐based drug might be of interest in cancer therapy. In the present study, the in vitro platination of the BRCA1 RING protein by the anticancer drug cisplatin was observed. The protein contained a preformed structure in the apo form with structural changes and resistance to limited proteolysis after Zn²⁺ binding. SDS‐PAGE and mass‐spectrometric analyses revealed that cisplatin preferentially formed monofunctional and bifunctional BRCA1 adducts. Tandem mass spectrometry (MS/MS) of the 656.29²⁺ ion indicated that the ion arose from [Pt(NH₃)₂(OH)]⁺ bound to the BRCA1 peptide ¹¹¹ENNSPEHLK¹¹⁹. The product‐ion spectrum revealed the Pt‐binding site on His117. Circular dichroism showed that the apo form, not holo form, of BRCA1 underwent more folded structural rearrangement upon cisplatin binding. Cisplatin‐bound protein exhibited an enhanced thermostability by 13°, resulting from the favorably intermolecular cross‐links driven by the free energy. Our findings demonstrated the first conformational and thermal evidences for a direct binding of cisplatin to the BRCA1 RING domain and could raise a possibility of selectively targeted treatment of cancer with less toxicity or improved response to conventional regimens.     

HTSDSF Explorer,A Novel Tool to Analyze High-throughput DSF Screenings

The identification of new drugs for novel therapeutic targets requires the screening of libraries containing tens of thousands of compounds. While experimental screenings are assisted by high-throughput technologies, in target-based biophysical assays, such as differential scanning fluorimetry (DSF), the analysis steps must be calculated manually, often combining several software packages. To simplify the determination of the melting temperature (T_m) of the target and the change induced by ligand binding (ΔT_m), we developed the HTSDSF explorer, a versatile, all-in-one, user-friendly application suite. Implemented as a server-client application, in the primary screenings, HTSDSF explorer pre-analyzes and displays the T_m and ΔT_m results interactively, thereby allowing the user to study hundreds of conditions and select the primary hits in minutes. This application also allows the determination of preliminary binding constants (K_D) through a series of subsequent dose–response assays on the primary hits, thereby facilitating the ranking of validated hits and the advance of drug discovery efforts.     

A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as K_D, k_on, k_off, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand–p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in T_m values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.     

Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB_69-333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB_69-333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB_69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.     

Structural Insights into Calcium-Bound S100P and the V Domain of the RAGE Complex

The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE) and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca²⁺-bound S100P using various biophysical techniques, including isothermal titration calorimetry (ITC), fluorescence spectroscopy, multidimensional NMR spectroscopy, functional assays and site-directed mutagenesis. The entropy-driven binding between the V domain of RAGE and Ca⁺²-bound S100P was found to lie in the micromolar range (K_d of ∼6 µM). NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases.     

High-Throughput 1,536-Well Fluorescence Polarization Assays for α1-Acid Glycoprotein and Human Serum Albumin Binding

Two major plasma proteins in humans are primarily responsible for drug binding, the α₁-acid-glycoprotein (AGP) and human serum albumin (HSA). The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP) of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC¹²⁸⁰ collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.     

DNA and glutathione interactions in cell-free media of asymmetric platinum(II) complexes cis- and trans-[PtCl2(isopropylamine)(1-methylimidazole)]: relations to their different antitumor effects

The global modification of mammalian and plasmid DNAs by the novel platinum compounds cis-[PtCl₂(isopropylamine)(1-methylimidazole)] and trans-[PtCl₂(isopropylamine)(1-methylimidazole)] and the reactivity of these compounds with reduced glutathione (GSH) were investigated in cell-free media using various biochemical and biophysical methods. Earlier cytotoxicity studies had revealed that the replacement of the NH₃ groups in cisplatin by the azole and isopropylamine ligands lowers the activity of cisplatin in both sensitive and resistant cell lines. The results of the present work show that this replacement does not considerably affect the DNA modifications by this drug, recognition of these modifications by HMGB1 protein, their repair, and reactivity of the platinum complex with GSH. These results were interpreted to mean that the reduced activity of this analog of cisplatin in tumor cell lines is due to factors that do not operate at the level of the target DNA. In contrast, earlier studies had shown that the replacement of the NH₃ groups in the clinically ineffective trans isomer (transplatin) by the azole and isopropylamine ligands results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin, which is distinctly different from that of cisplatin, but does not affect reactivity with GSH. Hence, the results of the present work are consistent with the view and support the hypothesis systematically tested by us and others that platinum drugs that bind to DNA in a fundamentally different manner from that of conventional cisplatin may have altered pharmacological properties.     

Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance

Membrane proteins (MPs) are prevalent drug discovery targets involved in many cell processes. Despite their high potential as drug targets, the study of MPs has been hindered by limitations in expression, purification and stabilization in order to acquire thermodynamic and kinetic parameters of small molecules binding. These bottlenecks are grounded on the mandatory use of detergents to isolate and extract MPs from the cell plasma membrane and the coexistence of multiple conformations, which reflects biochemical versatility and intrinsic instability of MPs. In this work ,we set out to define a new strategy to enable surface plasmon resonance (SPR) measurements on a thermostabilized and truncated version of the human adenosine (A_2A) G-protein-coupled receptor (GPCR) inserted in a lipid bilayer nanodisc in a label- and detergent-free manner by using a combination of affinity tags and GFP-based fluorescence techniques. We were able to detect and characterize small molecules binding kinetics on a GPCR fully embedded in a lipid environment. By providing a comparison between different binding assays in membranes, nanodiscs and detergent micelles, we show that nanodiscs can be used for small molecule binding studies by SPR to enhance the MP stability and to trigger a more native-like behaviour when compared to kinetics on A_2A receptors isolated in detergent. This work provides thus a new methodology in drug discovery to characterize the binding kinetics of small molecule ligands for MPs targets in a lipid environment.     

Guanidine-HCl Dependent Structural Unfolding of M-Crystallin: Fluctuating Native State Like Topologies and Intermolecular Association

Numerous experimental techniques and computational studies, proposed in recent times, have revolutionized the understanding of protein-folding paradigm. The complete understanding of protein folding and intermediates are of medical relevance, as the aggregation of misfolding proteins underlies various diseases, including some neurodegenerative disorders. Here, we describe the unfolding of M-crystallin, a βγ-crystallin homologue protein from archaea, from its native state to its denatured state using multidimensional NMR and other biophysical techniques. The protein, which was earlier characterized to be a predominantly β-sheet protein in its native state, shows different structural propensities (α and β), under different denaturing conditions. In 2 M GdmCl, the protein starts showing two distinct sets of peaks, with one arising from a partially unfolded state and the other from a completely folded state. The native secondary structural elements start disappearing as the denaturant concentration approaches 4 M. Subsequently, the protein is completely unfolded when the denaturant concentration is 6 M. The ¹⁵N relaxation data (T₁/T₂), heteronuclear ¹H-¹⁵N Overhauser effects (nOes), NOESY data, and other biophysical data taken together indicate that the protein shows a consistent, gradual change in its structural and motional preferences with increasing GdmCl concentration.     

Elucidating the interaction of clofazimine with bovine liver catalase; a comprehensive spectroscopic and molecular docking approach

Nowadays, understanding of interface between protein and drugs has become an active research area of interest. These types of interactions provide structural guidelines in drug design with greater clinical efficacy. Thus, structural changes in catalase induced by clofazimine were monitored by various biophysical techniques including UV‐visible spectrometer, fluorescence spectroscopy, circular dichroism, and dynamic light scattering techniques. Increase in absorption spectra (UV‐visible spectrum) confers the complex formation between drug and protein. Fluorescence quenching with a binding constants of 2.47 × 10⁴ M⁻¹ revealed that clofazimine binds with protein. Using fluorescence resonance energy transfer, the distance (r ) between the protein (donor) and drug (acceptor) was found to be 2.89 nm. Negative Gibbs free energy change (ΔG °) revealed that binding process is spontaneous. In addition, an increase in α‐helicity was observed by far‐UV circular dichroism spectra by adding clofazimine to protein. Dynamic light scattering results indicate that topology of bovine liver catalase was slightly altered in the presence of clofazimine. Hydrophobic interactions are the main forces between clofazimine and catalase interaction as depicted by molecular docking studies. Apart from hydrophobic interactions, some hydrogen bonding was also observed during docking method. The results obtained from the present study may establish abundant in optimizing the properties of ligand‐protein mixtures relevant for numerous formulations.     

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS₁) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS₁ using robotic ligand binding assays. NTS₁ is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.     

Development of Systemic in vitro Evolution and Its Application to Generation of Peptide-Aptamer-Based Inhibitors of Cathepsin E

Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC₅₀. This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (P_i101; SCGG IIII SCIA) have half an inhibition activity (K_i; 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.     

Reaction of human metallothionein-3 with cisplatin and transplatin

Human metallothioneins, small cysteine- and metal-rich proteins, play an important role in the acquired resistance to platinum-based anticancer drugs. These proteins contain a M(II)₄(CysS)₁₁ cluster and a M(II)₃(CysS)₉ cluster localized in the α-domain and the β-domain, respectively. The noninducible isoform metallothionein-3 (Zn₇MT-3) is mainly expressed in the brain, but was found overexpressed in a number of cancer tissues. Since the structural properties of this isoform substantially differ from those of the ubiquitously occurring Zn₇MT-1/Zn₇MT-2 isoforms, the reactions of cis-diamminedichloridoplatinum(II) (cisplatin) and trans-diamminedichloridoplatinum(II) (transplatin) with human Zn₇MT-3 were investigated and the products characterized. A comparison of the reaction kinetics revealed that transplatin reacts with cysteine ligands of Zn₇MT-3 faster than cisplatin. In both binding processes, stoichiometric amounts of Zn(II) were released from the protein. Marked differences between the reaction rates of cisplatin and transplatin binding to Zn₇MT-3 and the formation of the Pt–S bonds suggest that the binding of both Pt(II) compounds is a complex process, involving at least two subsequent binding steps. The electrospray ionization mass spectrometry characterization of the products showed that whereas all ligands in cisplatin were replaced by cysteine thiolates, transplatin retained its carrier ammine ligands. The ¹¹³Cd NMR studies of Pt₁ ¹¹³Cd₆MT-3 revealed that cisplatin binds preferentially to the β-domain of the protein. The rates of reaction of cisplatin and transplatin with Zn₇MT-3 were much faster than those of cisplatin and transplatin with Zn₇MT-2. The biological consequences of a substantially higher reactivity of cisplatin toward Zn₇MT-3 than Zn₇MT-2 in the acquired resistance to platinum-based drugs are discussed.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

Background

Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding.

Results

In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces.

Conclusions

NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.     

Differential genotoxic effects of antitumor trans-[PtCl2(E-iminoether)2] and cisplatin in Escherichia coli

In the present work, we measured survival and the platinum on the genome after treatment of repair-proficient or repair-deficient Escherichia coli strains with trans-[PtCl₂(E-iminoether)₂] and compared these results with the effects of “classical” cisplatin. We found that toxicity of antitumor trans-[PtCl₂(E-iminoether)₂] in repair-deficient trains was much less than that of cisplatin. This markedly reduced toxicity was not a consequence of the reduced uptake or low levels of DNA binding in the bacteria cells but rather appeared to reflect DNA binding mode of this trans-platinum drug different from that of cisplatin.     

Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs

The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe₄S₄]²⁺ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts.