Applications of Biophysics in High-Throughput Screening Hit Validation

Over the past three decades nuclear magnetic resonance spectroscopy has been developed into a mature technique for the characterization of interactions of small molecule ligands with their corresponding protein and nucleic acid receptors. In fact, a significant number of industrial and academic laboratories employ NMR for screening small molecule compound collections for binding to defined macromolecular targets, thus potentially providing initial, low affinity hits for a fragment-based approach in the drug discovery process. NMR is also applied to interrogate hits obtained by high throughput screening using biochemical assays and by virtual screening methods, for their ability to physically interact with the target receptor. In favorable cases a variety of NMR-based methods can also provide essential information to validate the hit, rank the different hits according to affinity, and to structurally analyze the ligand-target complex, thus providing essential information for structure-based optimization and medicinal chemistry. In this review a comprehensive overview of the large variety of NMR methods to study interactions between small molecule ligands and macromolecular receptors is provided, summarizing the physico-chemical bases of the different receptor- and ligand-observed experiments. The application of these methods for compound library screening and hit validation, with special emphasis on their contribution to fragment-based drug discovery strategies, is illustrated by recent examples selected from the literature and work in my laboratory.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

Macrophage migration inhibitory factor (MIF) is a major mediator in innate immunity and inflammation and a potential therapeutic target in multiple inflammatory, infectious and autoimmune diseases including cancer. Current therapeutic strategies for targeting MIF focus on modulating its biological activities using anti-MIF neutralizing antibodies or developing inhibitors of its tautomerase activity. Although the identity of its natural substrate remains unknown, several small molecule inhibitors have been reported to be effective inhibitors of MIF tautomerase activity in vitro. All these inhibitors were identified by rational design and structure-activity studies based on the structure of the catalytic site and/or non physiologic substrates of MIF. The lack of suitable high-throughput screening (HTS) assays has hindered the screening of chemical libraries and discovery of more diverse inhibitors. Motivated by the goal of discovering diverse classes of MIF inhibitors that modulate MIF activity specific targeting of its biochemical, conformation and quaternary structure properties, both activity based endpoint and kinetic HTS assays were developed, optimized and performed on diverse set of libraries. Using the endpoint assay, out of 15440 compounds screened, twelve novel inhibitors of MIF's catalytic activity (∼ 0.1% hit rate) with IC50s in the range of 1.5 to 15.5 µM were identified and validated. The interaction site and mechanism of action of all these inhibitors were defined using structure activity studies and a battery of biochemical and biophysical methods, including mass spectrometry, light scattering, and NMR. The effect on MIF biological activities was also examined on MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Using the kinetic-based activity assay, we screened 80,000 small molecules and identified and validated thirteen novel inhibitors of MIF catalytic activity with inhibition constant (Ki,app) values ranging from 0.5 to 13 µM. According to the structure and potency some of these molecules could be of great therapeutic potential. The MIF inhibitors emerging from these studies could be divided into four classes: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1 which could be classified into 5 groups; 2) a novel class of catalytic site inhibitors, 3) a class of trimer destabilizing inhibitors. These findings demonstrate the potential of HTS assays as attractive means to identify novel classes of MIF inhibitors as lead drug candidates and/or chemical tools for elucidating the biochemical and structural bases underlying the multifunctionality of MIF's in health and disease. Having these molecules in hand, will advance our understanding of protein-protein interactions mechanism for MIF oligomerization and may help to elucidate the role of MIF enzymatic activity in health and disease.

Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches. With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding. SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin. As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin. A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen. Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin. Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds. A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays. All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.

Fragment-based approaches are now routinely applied for lead development in pharmaceutical drug research. Usually, a small but well selected library of low molecular weight compounds is pre-screened by biochemical or biophysical methods such as surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) or thermal shift assay; often followed for promising hit candidates by X-ray crystallography. We designed a small fragment library consisting of 364 compounds that is not strictly compliant to the otherwise often followed Astex rule of three for fragment library composition.[1] Thereafter, our library was validated on the pepsin-like aspartyl protease endothiapepsin, which serves as a model system for proteins that are involved in serious diseases such as malaria (plasmepsins), hypertension (renin) and Alzheimer's disease (ß-secretase) and therefore, is a valid target for further drug development. Due to the small size of fragments, they frequently exhibit only low affinity to the applied target protein and thus are often hard to detect in any screening approach, reflected in little overlap between different screening methods. After initial screening, we decided to validate the entire library by X-ray crystallography, which requires a steady supply of crystals, reproducible soaking conditions and a reliable setup at a synchrotron source, such as HZB BESSY II BL14.1 [2], preferably with some automation in initial data processing and refinement. A total hit rate greater than 10% was obtained, which will be compared to results from other screening methods. The resulting crystal structures will be discussed and provide an ideal basis for further lead development.

Being the standard solvent for preparing stock solutions of compounds for drug discovery, DMSO is always present in assay buffers in concentrations ranging from 0.1 % to 5 % (v/v). Even at the lowest concentrations, DMSO-containing solutions can have significant effects on individual proteins and possible pitfalls cannot be eliminated. Herein, we used two protein systems, the lysine methyltransferases G9a/KMT1 C and SETD8/KMT5 A, to study the effects of DMSO on protein stability and on the binding of the corresponding inhibitors, using different biophysical methods such as nano Differential Scanning Fluorimetry (nanoDSF), Differential Scanning Fluorimetry (DSF), microscale thermophoresis (MST), and surface plasmon resonance (SPR), all widely used in drug discovery screening campaigns. We demonstrated that the effects of DMSO are protein- and technique-dependent and cannot be predicted or extrapolated on the basis of previous studies using different proteins and/or different assays. Moreover, we showed that the application of orthogonal biophysical methods can lead to different binding affinity data, thus confirming the importance of using at least two different orthogonal assays in screening campaigns. This variability should be taken into account in the selection and characterization of hit compounds, in order to avoid data misinterpretation.

Discovering small-molecule chemical probes of protein function has great potential to elucidate biological pathways and to provide early-stage proof-of-concept for target validation. Discovery of such probes therefore underpins many of the chemical biology and drug discovery efforts in both academia and the pharmaceutical industry. The process generally begins with screening small molecules to identify bona fide "hits" that bind non-covalently to a target protein. This chapter is concerned with the application of biophysical and structural techniques to small-molecule ligand screening, and with the validation of hits from both structural (binding mode) and energetic (binding affinity) stand-points. The methods discussed include differential scanning fluorimetry (thermal shift), fluorescence polarization (FP), surface plasmon resonance, ligand-observed NMR spectroscopy, isothermal titration calorimetry, and protein X-ray crystallography. The principles of these techniques and the fundamental nature of the observables used to detect macromolecule-ligand binding are briefly outlined. The practicalities, advantages, and disadvantages of each technique are described, particularly in the context of detecting weak affinities, as relevant to fragment screening. Fluorescence-based methods, which offer an attractive combination of high throughput and low cost are discussed in detail. It is argued that applying a combination of different methods provides the most robust and effective way to identify high-quality starting points for follow-up medicinal chemistry and to build structure-activity relationships that better inform effective development of high-quality, cell-active chemical probes by structure-based drug design.

High throughput screening (HTS) has become the cornerstone of lead identification for small molecules in drug discovery during the last quarter century. The evolution of the sciences and technologies that have evolved as the foundation of modern HTS campaigns are complex and require multidisciplinary interactions. Innovations in integrated automated systems, reagent systems enabled by molecular biology, computational capabilities, and visualization tools have converged to provide sophisticated tools to HTS practitioners. The success of HTS in an organization does not rest solely with those performing HTS but is critically dependent on the interactions of biology and chemistry members of the multidisciplinary teams throughout the early discovery process. Thus a basic knowledge and understanding of the components and processes of HTS is a necessary requirement for effective communication in planning, executing, and analyzing an HTS campaign. This chapter addresses the key components of HTS campaigns, common approaches, and related issues that should be understood by those engaged in small molecule drug discovery.

Fragment-based screening is an emerging technology which is used as an alternative to high-throughput screening (HTS), and often in parallel. Fragment screening focuses on very small compounds. Because of their small size and simplicity, fragments exhibit a low to medium binding affinity (mM to µM) and must therefore be screened at high concentration in order to detect binding events. Since some issues are associated with high-concentration screening in biochemical assays, biophysical methods are generally employed in fragment screening campaigns. Moreover, these techniques are very sensitive and some of them can give precise information about the binding mode of fragments, which facilitates the mandatory hit-to-lead optimization. One of the main advantages of fragment-based screening is that fragment hits generally exhibit a strong binding with respect to their size, and their subsequent optimization should lead to compounds with better pharmacokinetic properties compared to molecules evolved from HTS hits. In other words, fragments are interesting starting points for drug discovery projects. Besides, the chemical space of low-complexity compounds is very limited in comparison to that of drug-like molecules, and thus easier to explore with a screening library of limited size. Furthermore, the "combinatorial explosion" effect ensures that the resulting combinations of interlinked binding fragments may cover a significant part of "drug-like" chemical space. In parallel to experimental screening, virtual screening techniques, dedicated to fragments or wider compounds, are gaining momentum in order to further reduce the number of compounds to test. This article is a review of the latest news in both experimental and in silico virtual screening in the fragment-based discovery field. Given the specificity of this journal, special attention will be given to fragment library design.

Surface plasmon resonance (SPR) is a powerful technology that allows an information-rich assessment of the interactions of tethered protein molecular targets with their interacting ligands in real time. As such, SPR has proven useful at all stages of drug discovery, from initial hit finding (particularly the screening of fragment libraries) to mechanistic kinetic and thermodynamic profiling during hit validation and lead optimisation phases. Each of these stages of drug discovery involves somewhat different considerations in the optimal design and interpretation of SPR experiments. These factors will be discussed along with other relevant applications of SPR technology for drug discovery, such as in DMPK and toxicology assessments. Finally, the future developments in SPR for drug discovery will be considered.

AU‐rich elements (ARE) exist in the 3′‐untranslated regions of the mRNA transcribed from cell growth‐related genes such as proto‐oncogenes, cyclin‐related genes, and growth factors. HuR binds and stabilizes ARE‐mRNA. HuR is expressed abundantly in cancer cells and related malignant phenotypes. HuR knockdown attenuates the malignant phenotype of oral cancer cells. In this study, we screened 1570 compounds in the approved drug library by differential scanning fluorimetry (DSF) to discover a HuR‐targeted compound. Firstly, 55 compounds were selected by DSF. Then, 8 compounds that showed a shift in the melting temperature value in a concentration‐dependent manner were selected by DSF. Of them, suramin, an anti‐trypanosomal drug, binds to HuR, exhibiting fast‐on and fast‐off kinetic behavior on surface plasmon resonance (SPR). We confirmed that suramin significantly decreased mRNA and protein expression of cyclin A2 and cyclin B1. The cyclin A2 and cyclin B1 mRNAs were destabilized by suramin. Furthermore, the motile and invasive activities of a tongue carcinoma cell line treated with suramin were markedly lower than those of control cells. The above findings suggest that suramin binds to HuR and inhibits its function. We also showed that the anticancer effects of suramin were caused by the inhibition of HuR function, indicating its potential as a novel therapeutic agent in the treatment of oral cancer. Our results suggest that suramin, via its different mechanism, may effectively suppress progressive oral cancer that cannot be controlled using other anticancer agents.

Protein kinases are important drug targets, yet specific inhibitors have been developed for only a fraction of the more than 500 human kinases. A major challenge in designing inhibitors for highly related kinases is selectivity. Unlike their non-covalent counterparts, covalent inhibitors offer the advantage of selectively targeting structurally similar kinases by modifying specific protein side chains, particularly non-conserved cysteines. Previously, covalent fragment screens yielded potent and selective inhibitors for individual kinases such as ERK1/2 but have not been applied to the broader kinome. Furthermore, many of the accessible cysteine positions have not been addressed so far. Here, we outline a generalizable approach to sample ATP-site cysteines with fragment-like covalent inhibitors. We present the development of a kinase-focused covalent fragment library and its systematic screening against a curated selection of 47 kinases, with 60 active site-proximal cysteines using LC/MS and differential scanning fluorimetry (DSF) assays, followed by hit validation through various complementary techniques. Our findings expand the repertoire of targetable cysteines within protein kinases, provide insight into unique binding modes identified from crystal structures and deliver isoform-specific hits with promising profiles as starting points for the development of highly potent and selective covalent inhibitors.

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.

Validation of a high throughput screening assay to identify small molecules that target the eukaryotic replicative helicase.

The phosphatidylinositol 3-kinase (PI3K) pathway is involved in many cellular functions including cell growth, metabolism, and transformation. Hyperactivation of this pathway contributes to tumorigenesis, therefore, PI3K is a major target for anticancer drug discovery. Since the PI3Kα isoform is implicated mostly in cancer, we conducted a high-throughputscreening (HTS) campaign using a 3-step PI3K homogenous time-resolved fluorescence assay against this isoform bearing the H1047R mutation. A total of 288,000 synthetic and natural product-derived compounds were screened and of which, we identified 124 initial hits that were further selected by considering the predicted binding mode, relationship to known pan-assay interference compounds and previous descriptions as a lipid kinase inhibitor. A total of 24 compounds were then tested for concentration-dependent responses. These hit compounds provide novel scaffolds that can potentially be optimized to create novel PI3K inhibitors.