Discovery of Novel Allosteric Non‐Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding

Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti‐infective drug target. Currently available anti‐FPPS drugs are active‐site‐directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non‐bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure–activity relationships, and provide X‐ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none‐bone diseases.     

Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.     

Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N‐terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post‐translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.     

Structural Investigation of the Binding of 5‐Substituted Swainsonine Analogues to Golgi α‐Mannosidase II

Golgi α‐mannosidase II (GMII) is a key enzyme in the N‐glycosylation pathway and is a potential target for cancer chemotherapy. The natural product swainsonine is a potent inhibitor of GMII. In this paper we characterize the binding of 5α‐substituted swainsonine analogues to the soluble catalytic domain of Drosophila GMII by X‐ray crystallography. These inhibitors enjoy an advantage over previously reported GMII inhibitors in that they did not significantly decrease the inhibitory potential of the swainsonine head‐group. The phenyl groups of these analogues occupy a portion of the binding site not previously seen to be populated with either substrate analogues or other inhibitors and they form novel hydrophobic interactions. They displace a well‐organized water cluster, but the presence of a C(10) carbonyl allows the reestablishment of important hydrogen bonds. Already approximately tenfold more active against the Golgi enzyme than the lysosomal enzyme, these inhibitors offer the potential of being extended into the N‐acetylglucosamine binding site of GMII for the creation of even more potent and selective GMII inhibitors.     

Multi‐phosphorylation of the Intrinsically Disordered Unique Domain of c‐Src Studied by In‐Cell and Real‐Time NMR Spectroscopy

Intrinsically disordered regions (IDRs) are preferred sites for post‐translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase–phosphatase networks. Here we used real‐time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the “unique domain” of c‐Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin‐dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP‐dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK‐dependent sites, thus suggesting indirect effects of kinase–phosphatase networks. This study provides a proof‐of‐concept of the use of real‐time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains.     

X‐ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan–McDermid Syndrome

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan–McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X‐ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high‐resolution X‐ray structures of all CLKs, including the first CLK4 X‐ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX‐4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan‐CLK inhibition in PMDS.     

Rational Design of Domain‐Swapping‐Based c‐Type Cytochrome Heterodimers by Using Chimeric Proteins

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three‐dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c‐type cytochrome‐based chimeric proteins have been constructed and the domains swapped. According to X‐ray crystallographic analysis, the two chimeric proteins formed a domain‐swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H₂O coordinated hemes was formed. Binding of an oxygen molecule to the His/H₂O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe?O₂ stretching band was observed at 580 cm^?1 for the reduced/oxygenated heterodimer (at 554 cm^?1 under an ¹⁸O₂ atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.     

A Novel Tyrosine–Heme CO Covalent Linkage in F43Y Myoglobin: A New Post‐translational Modification of Heme Proteins

Heme post‐translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine–heme covalent C?O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4‐vinyl group. This highlights the diverse chemistry of heme post‐translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently‐bound heme proteins.     

Cyclic Hexapeptide Mimics of the LEDGF Integrase Recognition Loop in Complex with HIV‐1 Integrase

The p75 splice variant of lens epithelium‐derived growth factor (LEDGF) is a 75 kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre‐integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X‐ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV‐1 integrase core domain. Although the X‐ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low‐energy solution and bound conformations.     

para‐Substituted 2‐Phenyl‐3,4‐dihydroquinazolin‐4‐ones As Potent and Selective Tankyrase Inhibitors

Human tankyrases are attractive drug targets, especially for the treatment of cancer. We identified a set of highly potent tankyrase inhibitors based on a 2‐phenyl‐3,4‐dihydroquinazolin‐4‐one scaffold. Substitutions at the para position of the scaffold′s phenyl group were evaluated as a strategy to increase potency and improve selectivity. The best compounds displayed single‐digit nanomolar potencies, and profiling against several human diphtheria‐toxin‐like ADP‐ribosyltransferases revealed that a subset of these compounds are highly selective tankyrase inhibitors. The compounds also effectively inhibit Wnt signaling in HEK293 cells. The binding mode of all inhibitors was studied by protein X‐ray crystallography. This allowed us to establish a structural basis for the development of highly potent and selective tankyrase inhibitors based on the 2‐phenyl‐3,4‐dihydroquinazolin‐4‐one scaffold and outline a rational approach to the modification of other inhibitor scaffolds that bind to the nicotinamide site of the catalytic domain.     

Fragmenting the S100B–p53 Interaction: Combined Virtual/Biophysical Screening Approaches to Identify Ligands

S100B contributes to cell proliferation by binding the C terminus of p53 and inhibiting its tumor suppressor function. The use of multiple computational approaches to screen fragment libraries targeting the human S100B–p53 interaction site is reported. This in silico screening led to the identification of 280 novel prospective ligands. NMR spectroscopic experiments revealed specific binding at the p53 interaction site for a set of these compounds and confirmed their potential for further rational optimization. The X‐ray crystal structure determined for one of the binders revealed key intermolecular interactions, thus paving the way for structure‐based ligand optimization.     

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.     

Protein Contacts and Ligand Binding in the Inward‐Facing Model of Human P‐Glycoprotein

The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward‐open homology model of human P‐glycoprotein (P‐gp). The analysis revealed communication nets through hydrogen bonding in the NBD and at the NBD–TMD interface of each half involving residues from the adenosine triphosphate (ATP) motifs and the coupling helices of the intracellular loops. Similar networks have been identified in P‐gp conformations generated by molecular dynamics simulation. Differences have been recorded in the networking between both halves of P‐gp. Many of the residue contacts have also been observed in the X‐ray crystal structures of other ATP binding cassette (ABC) transporters, which confirms their validity. Next, possible binding pockets involving residues of importance for the TMD–NBD communication were identified. By studying these pockets, binding sites were suggested for rhodamine 123 (R‐site) and prazosin (regulatory site) at the NBD–TMD interface that agreed with the experimental data on their location. Additionally, one more R‐site in the protein cavity was proposed, in accordance with the available biochemical data. Together with the previously suggested Hoechst 33342 site (H‐site), all sites were interpreted with respect to their effects on the protein ATPase activity, in correspondence with the experimental observations. Several residues involved in key contacts in the P‐gp NBDs were proposed for further targeted mutagenesis experiments.     

Amino Acid‐Selective Segmental Isotope Labeling of Multidomain Proteins for Structural Biology

Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes up to several hundred kDa. However, the large molecular weight of proteins in such systems results in increased numbers of NMR signals, and the resulting spectral overlap typically imposes limitations. For multidomain proteins, segmental isotope labeling of individual domains facilitates the spectral interpretation by reducing the number of signals, but for large domains with small signal dispersion, signal overlap can persist. To overcome limitations arising from spectral overlap, we present a strategy that combines cell‐free expression and ligation of the expressed proteins to produce multidomain proteins with selective amino acid‐type labeling in individual domains. The bottleneck of intrinsically low cell‐free expression yields of precursor molecules was overcome by introducing new fusion constructs that allowed milligram production of ligation‐competent domains labeled in one or multiple amino acid types. Ligation‐competent unlabeled partner domains were produced in vivo, and subsequent domain ligation was achieved by using an on‐column strategy. This approach is illustrated with two multidomain RNA‐binding proteins, that is, the two C‐terminal RNA‐recognition motifs of the human polypyrimidine tract‐binding protein, and two highly homologous helix–turn–helix domains of the human glutamyl‐prolyl‐tRNA synthetase.     

Structure‐Based Design,Synthesis, and Evaluation of 2′‐(2‐Hydroxyethyl)‐2′‐deoxyadenosine and the 5′‐Diphosphate Derivative as Ribonucleotide Reductase Inhibitors

Analysis of the recently solved X‐ray crystal structures of Saccharomyces cerevisiae ribonucleotide reductase I (ScRnr1) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2′‐hydroxy group of the nucleoside ribose. In this study 2′‐(2‐hydroxyethyl)‐2′‐deoxyadenosine 1 and the 5′‐diphosphate derivative 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxymethylene group, to generate novel RNR inhibitors as potential antitumor agents. Herein we report the synthesis of analogues 1 and 2 , and the co‐crystal structure of adenosine diphosphate analogue 2 bound to ScRnr1, which shows the conserved water molecule is displaced as hypothesized.     

The High‐Pressure Characterization of Energetic Materials: 1‐Methyl‐5‐Nitramino‐1H‐Tetrazole

Pressure–volume relations and optical Raman and Infrared spectra of polycrystalline 1MNT have been obtained under quasi‐hydrostatic conditions up to 16 and 40 GPa, respectively, by using diamond anvil cell, synchrotron‐based angle‐resolved X‐ray diffraction, and microspectroscopy. The X‐ray measurements show that the pressure–volume relations remain smooth up to 16 GPa at room temperature, while vibrational measurements show no evidence of a phase transition to near 40 GPa. Anomalous increases of several vibrational intensities and bandwidths suggest that subtle molecular distortions and structural modifications occur in the crystal as pressure increases. Decompression experiments indicate the structural modifications are reversible.     

Inhibition of Eimeria tenella CDK‐Related Kinase 2: From Target Identification to Lead Compounds

Apicomplexan parasites encompass several human‐ and animal‐pathogenic protozoans such as Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella causes coccidiosis, a disease that afflicts chickens, leading to tremendous economic losses to the global poultry industry. The considerable increase in drug resistance makes it necessary to develop new therapeutic strategies against this parasite. Cyclin‐dependent kinases (CDKs) are key molecules in cell‐cycle regulation and are therefore prominent target proteins in parasitic diseases. Bioinformatics analysis revealed four potential CDK‐like proteins, of which one—E. tenella CDK‐related kinase 2 (EtCRK2)—has already been characterized by gene cloning and expression. 1 By using the CDK‐specific inhibitor flavopiridol in EtCRK2 enzyme assays and schizont maturation assays (SMA), we could chemically validate CDK‐like proteins as potential drug targets. An X‐ray crystal structure of human CDK2 (HsCDK2) served as a template to build protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP binding site between EtCRK2 and HsCDK2, as well as chicken CDK3, were addressed for the optimization of selective ATP‐competitive inhibitors. Virtual screening and “wet‐bench” high‐throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized, leading to a set of four promising lead compounds for development as EtCRK2 inhibitors.     

Three-dimensional pharmacophore design and biochemical screening identifies substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction

Protein interactions are increasingly appreciated as targets in small‐molecule drug discovery. The interaction between the adapter protein S100A10 and its binding partner annexin A2 is a potentially important drug target. To obtain small‐molecule starting points for inhibitors of this interaction, a three‐dimensional pharmacophore model was constructed from the X‐ray crystal structure of the complex between S100A10 and annexin A2. The pharmacophore model represents the favourable hydrophobic and hydrogen bond interactions between the two partners, as well as spatial and receptor site constraints (excluded volume spheres). Using this pharmacophore model, UNITY flex searches were carried out on a 3D library of 0.7 million commercially available compounds. This resulted in 568 hit compounds. Subsequently, GOLD docking studies were performed on these hits, and a set of 190 compounds were purchased and tested biochemically for inhibition of the protein interaction. Three compounds of similar chemical structure were identified as genuine inhibitors of the binding of annexin A2 to S100A10. The binding modes predicted by GOLD were in good agreement with their UNITY‐generated conformations. We synthesised a series of analogues revealing areas critical for binding. Thus computational predictions and biochemical screening can be used successfully to derive novel chemical classes of protein–protein interaction blockers.     

Identification of Green Tea Catechins as Potent Inhibitors of the Polo‐Box Domain of Polo‐Like kinase 1

Polo‐like kinase 1 (PLK1) plays crucial functions in multiple stages of mitosis and is considered to be a potential drug target for cancer therapy. The functions of PLK1 are mediated by its N‐terminal kinase domain and C‐terminal polo‐box domain (PBD). Most inhibitors targeting the kinase domain of PLK1 have a selectivity issue because of a high degree of structural conservation within kinase domains of all protein kinases. Here, we combined virtual and experimental screenings to identify green tea catechins as potent inhibitors of the PLK1 PBD. Initially, (?)‐epigallocatechin, one of the main components of green tea polyphenols, was found to significantly block the binding of fluorescein‐labeled phosphopeptide to the PBD at a concentration of 10 μm. Next, additional catechins were evaluated for their dose‐dependent inhibition of the PBD and preliminary structure–activity relationships were derived. Cellular analysis further showed that catechins interfere with the proper subcellular localization of PLK1, lead to cell‐cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types, such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa). Our data provides new insight into understanding the anticancer activities of green tea catechins.     

Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding

The impact of the incorporation of a non‐natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post‐translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X‐ray crystallography. The structure and dynamics of the apo state of AZH‐modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH‐modified PDZ3 and an azulene‐linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene‐linked peptide and the isotopically labelled protein. Co‐crystallisation and soaking failed for the peptide‐bound holo complex. NMR spectroscopy, however, allowed determination of the protein–ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA‐modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target.