Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies.

The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.

Modulation of Host-Parasite Interactions with Small Molecules Targeting Schistosoma mansoni microRNAs.

Parasites use different strategies of communication with their hosts. One communication channel that has been studied in recent years is the use of vesicle microRNAs to influence the host immune system by trematodes. sma-microRNA-10, secreted from Schistosoma mansoni, has been shown to influence the fate of host T-cells through manipulation of the NF-κB pathway. We have identified low molecular weight tool compounds that can interfere with this microRNA-mediated manipulation of the host immune system. We used a fragment-based screening approach by means of nuclear magnetic resonance (NMR) to identify binders to the precursor of the parasite sma-microRNA-10 present in their extracellular vesicles. The small fragments identified were used to select larger molecules. These molecules were shown to counteract the inhibition of NF-κB activity by sma-microRNA-10 in cell-based assays.

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

19 F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.

Discovery of Small Molecule Drugs Targeting the Biogenesis of microRNA-155 for the Treatment of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease that often leads to functional disorder in multiple organs, most often with symptoms related to skin lesions, cardiovascular disease and kidney damage. Although significant efforts have been made to find efficient therapies, it still remains uncured. Furthermore, the current therapy is often associated with adverse side effects and leads to a high economic burden for society. At Saverna Therapeutics, in collaboration with our partners, we initiated a lead discovery program that aims to modulate the biogenesis of miR-155. This non-coding RNA is upregulated in SLE patients and SLE mouse models. We used our drug discovery platform based on iterative fragment-based screening by nuclear magnetic resonance (NMR) and machine learning to identify ligands of pre-miR-155. After several iterations and chemical modifications, we have identified compounds that show structure-activity relationships in cellular assays. These inhibitors reduced the level of miR-155 as well as its associated inflammatory protein TNF α whereas the cells remained viable during exposure of the compounds.

NMR quality control of fragment libraries for screening.

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process.

How to Computationally Stack the Deck for Hit-to-Lead Generation: In Silico Molecular Interaction Energy Profiling for de Novo siRNA Guide Strand Surrogate Selection.

The Argonaute-2 protein is part of the RNA-induced silencing complex (RISC) and anchors the guide strand of the small interfering RNA (siRNA). The 3'-end of the RNA contains two unpaired nucleotides (3'-overhang) that interact with the PAZ (PIWI/Argonaute/Zwille) domain of the protein. Theoretical and experimental evidence points toward a direct connection between the PAZ/3'-overhang binding affinity and siRNA's potency and specificity. Among the challenges to overcome when deploying siRNA molecules as therapeutics are their ready degradation under physiological conditions and off-target effects. It has been demonstrated that nuclease resistance can be improved via replacement of the dinucleotide overhang by small molecules which retain the interactions of the RNA guide strand with the PAZ domain. Most commonly, nucleotide analogues are used to substitute the siRNA overhang. However, in this study we adopt a de novo approach to its modification. The X-ray structure of human Argonaute-2 PAZ domain served to perform virtual screening and molecular interaction energy profiling (i.e., decomposition of the force field calculated protein-ligand interaction energies) of tailored-to-purpose fragment libraries. The binding of fragments to the PAZ domain was validated experimentally by NMR spectroscopy. The in silico guided protocol led to the efficient discovery of a number of PAZ domain ligands with affinities comparable to that of a reference dinucleotide (UpU, Kd = 33 µM). Originally starting from a generic fragment library, hits progress from 930 µM down to 14 µM within three iterations for the fragments selected via in silico molecular interaction energy profiling from a bespoke library. These dinucleotide siRNA guide strand surrogates represent potential new siRNA-based therapeutics (when attached to siRNA to form bioconjugates) featuring improved efficacy, specificity, stability, and cellular uptake. This project yielded a portfolio of seven patent applications, four of which have been granted to date.

SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Characterization of the mechanism of action of the pan class I PI3K inhibitor NVP-BKM120 across a broad range of concentrations.

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G(2)-M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 µmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

Lipid-protein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane proteins.

The choice of a suitable detergent-based membrane mimetic is of crucial importance for high-resolution NMR studies of membrane proteins. The present report describes a new approach of detergent screening. It is based on the comparison of 2D (1)H,(15)N-correlation spectra of a protein in a membrane-bilayer "reference" medium and in "trial" detergent-based environments. The proposed "reference" medium is the lipid-protein nanodisc (LPN) representing nanoscale phospholipid bilayers wrapped around by apolipoprotein A-1. The set of zwitterionic (DPC, DMPC/DHPC), anionic (SDS, LMPG, LPPG), and weakly cationic (LDAO) detergent-based media was screened for their ability to represent the native structure of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP. The VSD/LPN complexes composed of saturated zwitterionic (DMPC), anionic (DMPG), or a mixture of unsaturated differently charged (POPC/DOPG, 3:1) lipids were used as reference. All assayed detergent media demonstrate similar CD spectra of the domain with a high level (approximately 60%) of overall helicity but different 2D NMR spectra. Using the reference spectrum of the VSD in LPN, we were able to choose the detergent composition in which the membrane-like structure of the VSD is preserved.

NMR structural and dynamical investigation of the isolated voltage-sensing domain of the potassium channel KvAP: implications for voltage gating.

The structure and dynamics of the isolated voltage-sensing domain (VSD) of the archaeal potassium channel KvAP was studied by high-resolution NMR. The almost complete backbone resonance assignment and partial side-chain assignment of the (2)H,(13)C,(15)N-labeled VSD were obtained for the protein domain solubilized in DPC/LDAO (2:1) mixed micelles. Secondary and tertiary structures of the VSD were characterized using secondary chemical shifts and NOE contacts. These data indicate that the spatial structure of the VSD solubilized in micelles corresponds to the structure of the domain in an open state of the channel. NOE contacts and secondary chemical shifts of amide protons indicate the presence of tightly bound water molecule as well as hydrogen bond formation involving an interhelical salt bridge (Asp62-R133) that stabilizes the overall structure of the domain. The backbone dynamics of the VSD was studied using (15)N relaxation measurements. The loop regions S1-S2 and S2-S3 were found mobile, while the S3-S4 loop (voltage-sensor paddle) was found stable at the ps-ns time scale. The moieties of S1, S2, S3, and S4 helices sharing interhelical contacts (at the level of the Asp62-R133 salt bridge) were observed in conformational exchange on the micros-ms time scale. Similar exchange-induced broadening of characteristic resonances was observed for the VSD solubilized in the membrane of lipid-protein nanodiscs composed of DMPC, DMPG, and POPC/DOPG lipids. Apparently, the observed interhelical motions represent an inherent property of the VSD of the KvAP channel and can play an important role in the voltage gating.

Time efficient detection of protein-ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment.

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3-5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns.

NMR-Based Strategies to Elucidate Bioactive Conformations of Weakly Binding Ligands.

Key processes in molecular biology are regulated by interactions between biomolecules. Protein-proteinand protein-ligand interactions, e.g., in signal transduction pathways, rely on the subtle interactionsbetween atoms at the binding interface of the involved molecules. Because biomolecules often havemany interacting partners, these interactions are not necessarily strong. The study of molecularrecognition gives insight into the complex network of signaling in life and is the basis of structure-baseddrug design.In the situation where the interaction is weak, one of the traditional methods that can be appliedto obtain structural information (internuclear distances) of the bound ligand is the so-called transferredNOE (trNOE) method. Recently, it became possible to use transferred cross-correlated relaxation (trCCR)to directly measure dihedral angles. The combined use of these two techniques significantly improvesthe precision of the structure determination of ligands weakly bound to macromolecules.The application of these techniques will be discussed in detail for a peptide derived fromIKKß bound to the protein NEMO that plays an important rolein the NFκB pathway.

Modulation of the Bioactive Conformation of Transforming Growth Factor ß: Possible Implications of Cation Binding for Biological Function.

In any organism, very precisely adjusted interaction and exchange of information between cellsis continuously required. These cooperative interactions involve numerous cytokines, acting throughcorresponding sets of cell-surface receptors. The transforming growth factor ß (TGF-ß)superfamily includes a variety of structurally related multifunctional cytokines that play criticalroles in maintaining cellular homeostasis and controlling cell fate. Response of a cell to a specificsignal it receives should depend upon the current state of the environment, including concentrationsof biologically relevant ions. One of the most biologically active ions, calcium, acts upon a specificcalcium signaling system that operates over a wide temporal range and regulates many cellularprocesses in continuous "cross-talk" with the TGF-ß signaling system. In additionto that, the structural and dynamical properties of TGF-ß molecules, along with detected directinteraction of them with the biologically relevant cations suggest another level of fine regulationof TGF-ß activity. The fact that both mono- and divalent cations bind in the same low-affinitysites implies that some competition of cations for interaction with TGF-ß can also occur in vivo,contributing to the diversity of TGF-ß biological functions.

Crystal structure of human estrogen-related receptor alpha in complex with a synthetic inverse agonist reveals its novel molecular mechanism.

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).