Zebrafish bioassay-guided microfractionation for the rapid in vivo identification of pharmacologically active natural products

The rapid acquisition of structural and bioactivity information on natural products (NPs) at the sub- milligram scale is key for performing efficient bioactivity-guided isolations. Zebrafish offer the possibility of rapid in vivo bioactivity analysis of small molecules at the microgram scale - an attractive feature when combined with high-resolution fractionation technologies and analytical methods such as UHPLC-TOF-MS and microflow NMR. Numerous biomedically relevant assays are now available in zebrafish, encompassing most indication areas. Zebrafish also provide the possibility to screen bioactive compounds for potential hepato-, cardio-, and neurotoxicities at a very early stage in the drug discovery process. Here we describe two strategies using zebrafish bioassays for the high-resolution in vivo bioactivity profiling of medicinal plants, using either a one-step or a two-step procedure for active compound isolation directly into 96-well plates. The analysis of the microfractions by microflow NMR in combination with UHPLC-TOF-MS of the extract enables the rapid dereplication of compounds and an estimation of their microgram quantities for zebrafish bioassays. Both the one-step and the two-step isolation procedures enable a rapid estimation of the bioactive potential of NPs directly from crude extracts. In summary, we present an in vivo , microgram-scale NP discovery platform combining zebrafish bioassays with microscale analytics to identify, isolate and evaluate pharmacologically active NPs.     

Discovering small-molecule estrogen receptor α/coactivator binding inhibitors: high-throughput screening, ligand development, and models for enhanced potency

Small molecules, namely coactivator binding inhibitors (CBIs), that block estrogen signaling by directly inhibiting the interaction of the estrogen receptor (ER) with coactivator proteins act in a fundamentally different way to traditional antagonists, which displace the endogenous ligand estradiol. To complement our prior efforts at CBI discovery by de?novo design, we used high-throughput screening (HTS) to identify CBIs of novel structure and subsequently investigated two HTS hits by analogue synthesis, finding many compounds with low micromolar potencies in cell-based reporter gene assays. We examined structure-activity trends in both series, using induced-fit computational docking to propose binding poses for these molecules in the coactivator binding groove. Analysis of the structure of the ER-steroid receptor coactivator (SRC) complex suggests that all four hydrophobic residues within the SRC nuclear receptor box sequence are important binding elements. Thus, insufficient water displacement upon binding of the smaller CBIs in the expansive complexation site may be limiting the potency of the compounds in these series, which suggests that higher potency CBIs might be found by screening compound libraries enriched with larger molecules.     

Computer-aided evaluation and exploration of chemical spaces constrained by reaction pathways

The processes of molecular design and synthetic route selection are necessarily intertwined during discovery. Computational tools have been developed to facilitate synthesis planning, but in a discovery setting, finding a single route to a single molecule of interest may be less important than finding a route that enables rapid access to a library of analogs. Here, we demonstrate how we can estimate route “diversifiability” and use it as a criterion during route selection. We illustrate how the chemical space of synthetically accessible analogs is influenced by properties of alternative starting materials or constraints on their cost. Finally, we integrate these analyses with a synthesizability-constrained hit expansion workflow in a virtual screening pipeline for focused library expansion around putative hits to support molecular optimization. As medicinal chemistry and adjacent fields shift toward more autonomous design and synthesis of new molecules, it will be increasingly important to embed considerations of synthesizability into molecular design to ensure that computational recommendations are actionable.     

Using a Fragment‐Based Approach To Target Protein–Protein Interactions

The ability to identify inhibitors of protein–protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment‐based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment‐based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X‐ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein–protein interaction site.     

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-typeplasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimideleads to a decrease in affinity for lysine and fibrin, to adecrease of plasminogen activation activity in the presenceof a fibrin mimic, but leaves amidolytic activity and plasminogenactivation without fibrin mimic unaffected. Experiments withkringle-2 ligands and a deletion mutant of t-PA (K₂P) suggeststhat glutamic or aspartic acid residues in K₂ of t-PA are involvedin stimulation of activity, lysine binding and fibrin binding.Mutant t-PA molecules were constructed by site-directed mutagenesisin which one or two of the five aspartic or glutamic acid residuesin K₂ were changed to asparagine or glutamine respectively.Mutation of Asp236 and/or Asp238 leads to t-PA molecules with3- to 4-fold lower specific activity in the presence of fibrinmimic and having no detectable affinity for lysine analogs.However, fibrin binding was not influenced. Mutation of Glu254also leads to a 3- to 4-fold lower activity, but to a much smallerreduction of lysine or fibrin binding. Residues Asp236 and Asp238are both essential for binding to lysine derivatives, whileGlu254 might be involved but is not essential. Residues Asp236,Asp238 and Glu254 are all three involved in stimulation of activity.Remarkably, mutation of residues Asp236 and/or Asp238 appearsnot to influence fibrin binding of t-PA whereas that of Glu254does.     

Concerted Electron/Proton Transfer Mechanism in the Oxidation of Phenols by Laccase

This study aimed to assess structural requirements in the enzyme/substrate interactions that are responsible for tuning the enzymatic reactivity. To better assess the role of the aspartic residue in the substrate‐binding pocket of basidiomycete‐type laccases, we compared the catalytic efficiency of wild‐type enzymes to that of a mutant in which carboxylic acid residue Asp206 was changed to alanine. Oxidation efficiency towards phenolic substrates by laccases of Trametes villosa, Trametes versicolor and a T. versicolor D206A mutant was studied at two pH values. By the Hammett approach and Marcus analysis, we obtained unambiguous evidence that the oxidation takes place by a concerted electron/proton transfer (EPT) mechanism, and that at pH 5 (optimum pH for enzyme activity) the phenolic proton is transferred to Asp206 during the concerted electron/proton transfer process.     

A Focus on Unusual ECL2 Interactions Yields β2-Adrenergic Receptor Antagonists with Unprecedented Scaffolds

The binding pockets of aminergic G protein-coupled receptors are often targeted by drugs and virtual screening campaigns. In order to find ligands with unprecedented scaffolds for one of the best-investigated receptors of this subfamily, the β₂-adrenergic receptor, we conducted a docking-based screen insisting that molecules would address previously untargeted residues in extracellular loop 2. We here report the discovery of ligands with a previously undescribed coumaran-based scaffold. Furthermore, we provide an analysis of the added value that X-ray structures in different conformations deliver for such docking screens.     

Chemically Synthesized 58‐mer LysM Domain Binds Lipochitin Oligosaccharide

Recognition of carbohydrates by proteins is a ubiquitous biochemical process. In legume–rhizobium symbiosis, lipochitin oligosaccharides, also referred to as nodulation (nod) factors, function as primary rhizobial signal molecules to trigger root nodule development. Perception of these signal molecules is receptor mediated, and nod factor receptor 5 (NFR5) from the model legume Lotus japonicus is predicted to contain three LysM domain binding sites. Here we studied the interactions between nod factor and each of the three NFR5 LysM domains, which were chemically synthesized. LysM domain variants (up to 58 amino acids) designed to optimize solubility were chemically assembled by solid‐phase peptide synthesis (SPPS) with microwave heating. Their interaction with nod factors and chitin oligosaccharides was studied by isothermal titration calorimetry and circular dichroism (CD) spectroscopy. LysM2 showed a change in folding upon nod factor binding, thus providing direct evidence that the LysM domain of NFR5 recognizes lipochitin oligosaccharides. These results clearly show that the L. japonicus LysM2 domain binds to the nod factor from Mesorhizobium loti, thereby causing a conformational change in the LysM2 domain. The preferential affinity for nod factors over chitin oligosaccharides was demonstrated by a newly developed glycan microarray. Besides the biological implications, our approach shows that carbohydrate binding to a small protein domain can be detected by CD spectroscopy.     

Multiparameter Kinetic Analysis for Covalent Fragment Optimization by Using Quantitative Irreversible Tethering (qIT)

Chemical probes that covalently modify cysteine residues in a protein-specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data-driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure–activity relationships through the simultaneous determination of K_i, k_inact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile-sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment-merging strategy.     

Library Screening by Means of Mass Spectrometry (MS) Binding Assays-Exemplarily Demonstrated for a Pseudostatic Library Addressing γ-Aminobutyric Acid (GABA) Transporter 1 (GAT1)

In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ‐aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine‐3‐carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays—the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1‐{2‐[2′‐(1,1’‐biphenyl‐2‐ylmethylidene)hydrazine]ethyl}piperidine‐3‐carboxylic acid and 1‐(2‐{2′‐[1‐(2‐thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine‐3‐carboxylic acid, were found to be potent GAT1 ligands exhibiting pK_i values of 6.186±0.028 and 6.229±0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including membrane‐bound targets such as G protein coupled receptors (GPCRs), ion channels and transporters. As such, this strategy displays high potential in the drug discovery process.     

Bacterially Secretable Single-Chain Tandem Macrocyclic Peptides for High Affinity and Inhibitory Activity

The inhibition of protein-protein interactions (PPIs) is an effective approach for therapy. Owing to their large binding surface areas to target proteins, macrocyclic peptides are suitable molecules for PPI inhibition. In this study, we developed single-chain tandem macrocyclic peptides (STaMPtides) that inhibits the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). They were artificially designed to comprise two different VEGFR2-binding macrocyclic peptides linked in tandem by peptide linkers and secreted by Corynebacterium glutamicum. Most potent VEGFR2-inhibitory STaMPtides with length-optimized linkers exhibited >1000 times stronger inhibitory activity than their parental monomeric peptides, possibly due to the avidity effect of heterodimerization. Our approach of using STaMPtides for PPI inhibition may be used to inhibit other extracellular factors, such as growth factors and cytokines.     

Prediction of the 3D structure of FMRF-amide neuropeptides bound to the mouse MrgC11 GPCR and experimental validation

We report the 3D structure predicted for the mouse MrgC11 (mMrgC11) receptor by using the MembStruk computational protocol, and the predicted binding site for the F-M-R-F-NH(2) neuropeptide together with four singly chirally modified ligands. We predicted that the R-F-NH(2) part of the tetrapeptide sticks down into the protein between the transmembrane (TM) domains 3, 4, 5, and 6. The Phe (F-NH(2)) interacted favorably with Tyr110 (TM3), while the Arg makes salt bridges to Asp161 (TM4) and Asp179 (TM5). We predicted that the Met extends from the binding site, but the terminal Phe residue sticks back into an aromatic/hydrophobic site flanked by Tyr237, Leu238, Leu240, and Tyr256 (TM6), and Trp162 (TM4). We carried out subsequent mutagenesis experiments followed by intracellular calcium-release assays that demonstrated the dramatic decrease in activity for the Tyr110Ala, Asp161Ala, and Asp179Ala substitutions, which was predicted by our model. These experiments provide strong evidence that our predicted G protein-coupled receptor (GPCR) structure is sufficiently accurate to identify binding sites for selective ligands. Similar studies were made with the mMrgA1 receptor, which did not bind the R-F-NH(2) dipeptide; we explain this to be due to the increased hydrophobic character of the binding pocket in mMrgA1.     

Exploration of Ligand-receptor Binding and Mechanisms for Alginate Reduction and Phenotype Reversion by Mucoid Pseudomonas aeruginosa

Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3’,5’)-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.     

Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3

Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell‐based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium‐labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [³H]N‐{1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐2‐[4‐fluoro‐3‐(trifluoromethyl)phenyl]‐N‐[(1‐methylpiperidin‐4‐yl)methyl]acetamide). RAMX3 is chemically derived from 8‐azaquinazolinone‐type allosteric modulators and binds to the CXCR3 receptor with a K_d value of 1.08 nM (specific activity: 80.4 Ci mmol^?1). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8‐azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.     

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.     

Anti‐HIV Small‐Molecule Binding in the Peptide Subpocket of the CXCR4:CVX15 Crystal Structure

The CXC chemokine receptor 4 (CXCR4) is involved in chemotaxis and serves as a coreceptor for T‐tropic HIV‐1 viral entry, thus making this receptor an attractive drug target. Recently, crystal structures of CXCR4 were reported as complexes with the small molecule IT1t and the CVX15 peptide. Follow‐up efforts to model different antagonists into the small molecule CXCR4:IT1t crystal structure did not generate poses consistent with either the X‐ray crystal structure or site‐directed mutagenesis (SDM). Here, we compare the binding pockets of the two CXCR4 crystal structures, revealing differences in helices IV, V, VI, and VII, with major differences for the His203 residue buried in the binding pocket. The small molecule antagonist AMD11070 was docked into both CXCR4 crystal structures. An AMD11070 pose identified from the CXCR4:CVX15 model presented interactions with Asp171, Glu288, Trp94, and Asp97, consistent with published SDM data, thus suggesting it is the bioactive pose. A CXCR4 receptor model was optimized around this pose of AMD11070, and the resulting model correlated HIV‐1 inhibition with MM‐GBSA docking scores for a congeneric AMD11070‐like series. Subsequent NAMFIS NMR results successfully linked the proposed binding pose to an independent experimental structure. These results strongly suggest that not all small molecules will bind to CXCR4 in a similar manner as IT1t. Instead, the CXCR4:CVX15 crystal structure may provide a binding locus for small organic molecules that is more suitable than the secondary IT1t site. This work is expected to provide modeling insights useful for future CXCR4 antagonist and X4‐tropic HIV‐1 based drug design efforts.     

Experimental and computational active site mapping as a starting point to fragment-based lead discovery

Small highly soluble probe molecules such as aniline, urea, N‐methylurea, 2‐bromoacetate, 1,2‐propanediol, nitrous oxide, benzamidine, and phenol were soaked into crystals of various proteins to map their binding pockets and to detect hot spots of binding with respect to hydrophobic and hydrophilic properties. The selected probe molecules were first tested at the zinc protease thermolysin. They were then applied to a wider range of proteins such as protein kinase A, D ‐xylose isomerase, 4‐diphosphocytidyl‐2C‐methyl‐D ‐erythritol synthase, endothiapepsin, and secreted aspartic protease 2. The crystal structures obtained clearly show that the probe molecules populate the protein binding pockets in an ordered fashion. The thus characterized, experimentally observed hot spots of binding were subjected to computational active site mapping using HotspotsX. This approach uses knowledge‐based pair potentials to detect favorable binding positions for various atom types. Good agreement between the in silico hot spot predictions and the experimentally observed positions of the polar hydrogen bond forming functional groups and hydrophobic portions was obtained. Finally, we compared the observed poses of the small‐molecule probes with those of much larger structurally related ligands. They coincide remarkably well with the larger ligands, considering their spatial orientation and the experienced interaction patterns. This observation confirms the fundamental hypothesis of fragment‐based lead discovery: that binding poses, even of very small molecular probes, do not significantly deviate or move once a ligand is grown further into the binding site. This underscores the fact that these probes populate given hot spots and can be regarded as relevant seeds for further design.     

Targeting SARS-CoV-2 RBD Interface: a Supervised Computational Data-Driven Approach to Identify Potential Modulators

Coronavirus disease 2019 (COVID-19) has spread out as a pandemic threat affecting over 2 million people. The infectious process initiates via binding of SARS-CoV-2 Spike (S) glycoprotein to host angiotensin-converting enzyme 2 (ACE2). The interaction is mediated by the receptor-binding domain (RBD) of S glycoprotein, promoting host receptor recognition and binding to ACE2 peptidase domain (PD), thus representing a promising target for therapeutic intervention. Herein, we present a computational study aimed at identifying small molecules potentially able to target RBD. Although targeting PPI remains a challenge in drug discovery, our investigation highlights that interaction between SARS-CoV-2 RBD and ACE2 PD might be prone to small molecule modulation, due to the hydrophilic nature of the bi-molecular recognition process and the presence of druggable hot spots. The fundamental objective is to identify, and provide to the international scientific community, hit molecules potentially suitable to enter the drug discovery process, preclinical validation and development.     

Sulfotyrosine recognition as marker for druggable sites in the extracellular space

Chemokine signaling is a well-known agent of autoimmune disease, HIV infection, and cancer. Drug discovery efforts for these signaling molecules have focused on developing inhibitors targeting their associated G protein-coupled receptors. Recently, we used a structure-based approach directed at the sulfotyrosine-binding pocket of the chemokine CXCL12, and thereby demonstrated that small molecule inhibitors acting upon the chemokine ligand form an alternative therapeutic avenue. Although the 50 members of the chemokine family share varying degrees of sequence homology (some as little as 20%), all members retain the canonical chemokine fold. Here we show that an equivalent sulfotyrosine-binding pocket appears to be conserved across the chemokine superfamily. We monitored sulfotyrosine binding to four representative chemokines by NMR. The results suggest that most chemokines harbor a sulfotyrosine recognition site analogous to the cleft on CXCL12 that binds sulfotyrosine 21 of the receptor CXCR4. Rational drug discovery efforts targeting these sites may be useful in the development of specific as well as broad-spectrum chemokine inhibitors.     

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution ¹⁹F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.