Conversion of methionine biosynthesis in Escherichia coli from trans- to direct-sulfurylation enhances extracellular methionine levels.

Methionine is an essential amino acid in mammals and a precursor for vital metabolites required for the survival of all organisms. Consequently, its inclusion is required in diverse applications, such as food, feed, and pharmaceuticals. Although amino acids and other metabolites are commonly produced through microbial fermentation, high-yield biosynthesis of L-methionine remains a significant challenge due to the strict cellular regulation of the biosynthesis pathway. As a result, methionine is produced primarily synthetically, resulting in a racemic mixture of D,L-methionine. This study explores methionine bio-production in E. coli by replacing its native trans-sulfurylation pathway with the more common direct-sulfurylation pathway used by other bacteria. To this end, we generated a methionine auxotroph E. coli strain (MG1655) by simultaneously deleting metA and metB genes and complementing them with metX and metY from different bacteria. Complementation of the genetically modified E. coli with metX/metY from Cyclobacterium marinum or Deinococcus geothermalis, together with the deletion of the global repressor metJ and overexpression of the transporter yjeH, resulted in a substantial increase of up to 126 and 160-fold methionine relative to the wild-type strain, respectively, and accumulation of up to 700 mg/L using minimal MOPS medium and 2 ml culture. Our findings provide a method to study methionine biosynthesis and a chassis for enhancing L-methionine production by fermentation.

Endotoxin-free gram-negative bacterium as a system for production and secretion of recombinant proteins.

Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in SJ identified using whole-genome computerized analysis, induced secretion of α Amylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80 µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines and as a live bacteria delivery system for protein vaccines. KEY POINTS: • Novel molecular tools for protein expression in non-model bacteria. • Bacteria with GSL instead of LPS as a potential vector for protein delivery.

Discovery and characterization of small molecule inhibitors of cystathionine gamma-synthase with in planta activity.

The synthesis of essential amino acids in plants is pivotal for their viability and growth, and these cellular pathways are therefore targeted for the discovery of new molecules for weed control. Herein, we describe the discovery and design of small molecule inhibitors of cystathionine gamma-synthase, a key enzyme in the biosynthesis of methionine. Based on in silico screening and filtering of a large molecular database followed by the in vitro selection of molecules, we identified small molecules capable of binding the target enzyme. Molecular modelling of the interaction and direct biophysical binding enabled us to explore a focussed chemical expansion set of molecules characterized by an active phenyl-benzamide chemical group. These molecules are bio-active and efficiently inhibit the viability of BY-2 tobacco cells and seedlings growth of Arabidopsis thaliana on agar plates.

Intracellular Protein-Drug Interactions Probed by Direct Mass Spectrometry of Cell Lysates.

Understanding protein-ligand interactions in a cellular context is an important goal in molecular biology and biochemistry, and particularly for drug development. Investigators must demonstrate that drugs penetrate cells and specifically bind their targets. Towards that end, we present a native mass spectrometry (MS)-based method for analyzing drug uptake and target engagement in eukaryotic cells. This method is based on our previously introduced direct-MS method for rapid analysis of proteins directly from crude samples. Here, direct-MS enables label-free studies of protein-drug binding in human cells and is used to determine binding affinities of lead compounds in crude samples. We anticipate that this method will enable the application of native MS to a range of problems where cellular context is important, including protein-protein interactions, drug uptake and binding, and characterization of therapeutic proteins.

Inhibition of PD1:PD-L1 interaction by an E. coli-derived optimized PD1 variant.

Immune-checkpoint receptors are a set of signal transduction proteins that can stimulate or inhibit specific anti-tumor responses. It is well established that cancer cells interact with different immune checkpoints to shut down T-cell response, thereby enabling cancer proliferation. Given the importance of immune checkpoint receptors, a structure-function analysis of these systems is imperative. However, recombinant expression and purification of these membrane originated proteins is still a challenge. Therefore, many attempts are being made to improve their expression and solubility while preserving their biological relevance. For this purpose, we designed an E. coli-based optimization system that enables the acquisition of mutations that increases protein solubility and affinity towards its native ligand, while maintaining biological activity. Here we focused on the well-characterized extracellular domain of the 'programmed cell death protein 1' (PD1), an immune checkpoint receptor known to inhibit T-cell proliferation by interacting with its ligands PD-L1 and PD-L2. The simple ELISA-based screening system shown here enabled the identification of high-affinity, highly soluble, functional variants derived from the extracellular domain of human PD1. The system was based on the expression of a GST-tagged variants library in E. coli, which enabled the selection of improved PD1 variants after a single optimization round. Within only two screening rounds, the most active variant showed a 5-fold higher affinity and 2.4-fold enhanced cellular activity as compared to the wild type protein. This scheme can be translated toward other types of challenging receptors toward development of research tools or alternative therapeutics.

An ELISA for the study of calcineurin-NFAT unstructured region interaction.

Calcineurin is a phosphatase that targets the transcription factor, nuclear factor of activated T-cells (NFAT) dephosphorylates multiple sites along NFAT's regulatory domain. The calcineurin-NFAT complex interaction is mediated through two conserved binding motifs known as the PxIxIT and LxVP, which are located at the N- and C- terminus to the phosphorylation sites. The vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the investigation of the structural aspects that govern their complex formation and in the discovery of protein-protein interaction inhibitors; the latter interfere with calcineurin-NFAT complex formation while keeping calcineurin's catalytic site free. To assist additional biophysical study of the calcineurin-NFAT structure-function relation and to screen for new inhibitors, we present a robust and cost-effective Enzyme Linked Immuno Sorbent Assay (ELISA) that is based on the interaction of calcineurin with the NFAT homology region. The latter includes the two calcineurin's binding sites, in addition to the phosphorylation sites. The ELISA experiment shown here can thus be applied towards the study of important structural aspects of the complex and for the discovery of new inhibitors. This will allow for a better understanding of T-cell activation switch.

Newcastle disease virus: is an updated attenuated vaccine needed?

Newcastle disease virus (NDV) is a major cause of infectious mortality and morbidity in poultry worldwide. It is an enveloped virus with two outer-membrane proteins-haemagglutinin-neuraminidase (HN) and fusion protein (F)-that induce neutralizing antibodies. All NDV strains belong to one serotype. Yet, NDV vaccines, derived from genotype II, do not fully prevent infection or shedding of viruses from other genotypes. The aim of this study was to test if an updated vaccine is required. For this purpose, NDVs isolated from infected, albeit heavily vaccinated, flocks were genetically and immunologically characterized. Amino acid differences in F and HN protein sequences were identified between the vaccine strain and each of the isolates, some specifically at the neutralization sites. Whereas all tested isolates showed similar haemagglutination-inhibition (HI) titres, 100-100,000 times higher antibody-to-virus ratios were needed to neutralize viral propagation in embryos by the field isolates versus the vaccine strain. As a result, a model and an equation were developed to explain the phenomenon of escape in one-serotype viruses and to calculate the HI values needed for protection, depending on variation rate at key positions. In conclusion, to confer full protection against NDVs that differ from the vaccine strain at the neutralizing epitopes, very high levels of antibodies should be raised and maintained to compensate for the reduction in the number of effective epitopes; alternatively, an adjusted attenuated vaccine should be developed-a task made possible in the current era of reverse vaccinology.

Discovering new mode-of-action pesticide leads inhibiting protein-protein interactions: example targeting plant O-acetylserine sulfhydrylase.

BACKGROUND: The widespread evolution of pesticide resistance poses a significant challenge to current agriculture, necessitating the discovery of molecules with new modes of action. Despite extensive efforts, no major molecules with new modes of action have been commercialized for decades. Most pesticides function by binding to specific pockets on target enzymes, enabling a single target site mutation to confer resistance. An alternative approach is the disruption of protein-protein interactions (PPI), which require complementary mutations on both interacting partners for resistance to occur. Thus, our aim is the discovery and design of small-molecule inhibitors that target the interface of the PPI complex of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT), key obligatory interacting plant enzymes involved in the biosynthesis of the amino acid cysteine. RESULTS: By employing in silico filtering techniques on a virtual library of 30 million small molecules, we identified initial hits capable of binding OASS and interfering with its interaction with a peptide derived from SAT with a half-maximal inhibitory concentration (IC50) of 34 µm. Subsequently, we conducted molecular chemical optimizations, generating an early lead molecule (PJ4) with an IC50 value of 4 µm. PJ4 successfully inhibited the germination of Arabidopsis thaliana seedlings and inhibited clover growth in a pre-emergence application at an effective concentration of 4.6 kg ha-1. CONCLUSION: These new compounds described herein can serve as promising leads for further optimization as herbicides with a new mode-of-action. This technology can be used for discovering new modes of action chemicals inhibiting all pest groups. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Application of In Silico Filtering and Isothermal Titration Calorimetry for the Discovery of Small Molecule Inhibitors of MDM2.

The initial discovery phase of protein modulators, which consists of filtering molecular libraries and in vitro direct binding validation, is central in drug discovery. Thus, virtual screening of large molecular libraries, together with the evaluation of binding affinity by isothermal calorimetry, generates an efficient experimental setup. Herein, we applied virtual screening for discovering small molecule inhibitors of MDM2, a major negative regulator of the tumor suppressor p53, and thus a promising therapeutic target. A library of 20 million small molecules was screened against an averaged model derived from multiple structural conformations of MDM2 based on published structures. Selected molecules originating from the computational filtering were tested in vitro for their direct binding to MDM2 via isothermal titration calorimetry. Three new molecules, representing distinct chemical scaffolds, showed binding to MDM2. These were further evaluated by exploring structure-similar chemical analogues. Two scaffolds were further evaluated by de novo synthesis of molecules derived from the initial molecules that bound MDM2, one with a central oxoazetidine acetamide and one with benzene sulfonamide. Several molecules derived from these scaffolds increased wild-type p53 activity in MCF7 cancer cells. These set a basis for further chemical optimization and the development of new chemical entities as anticancer drugs.

Protection against avian coronavirus conferred by oral vaccination with live bacteria secreting LTB-fused viral proteins.

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8+ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 107.5 EID50/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks.

Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine.

The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-γ and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs.

Conserved interactions required for inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands' interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50 µM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.

T-cell inactivation and immunosuppressive activity induced by HIV gp41 via novel interacting motif.

Fusion peptide (FP) of the HIV gp41 molecule inserts into the T cell membrane during virus-cell fusion. FP also blocks the TCR/CD3 interaction needed for antigen-triggered T cell activation. Here we used in vitro (fluorescence and immunoprecipitation), in vivo (T cell mediated autoimmune disease adjuvant arthritis), and in silico methods to identify the FP-TCR novel interaction motif: the alpha-helical transmembrane domain (TMD) of the TCR alpha chain, and the beta-sheet 5-13 region of the 16 N-terminal aa of FP (FP(1-16)). Deciphering the molecular mechanism of the immunosuppressive activity of FP provides a new potential target to overcome the immunosuppressant activity of HIV, and in addition a tool for down-regulating immune mediated inflammation.

Molecular flexibility in ab initio drug docking to DNA: binding-site and binding-mode transitions in all-atom Monte Carlo simulations.

The dynamics of biological processes depend on the structure and flexibility of the interacting molecules. In particular, the conformational diversity of DNA allows for large deformations upon binding. Drug-DNA interactions are of high pharmaceutical interest since the mode of action of anticancer, antiviral, antibacterial and other drugs is directly associated with their binding to DNA. A reliable prediction of drug-DNA binding at the atomic level by molecular docking methods provides the basis for the design of new drug compounds. Here, we propose a novel Monte Carlo (MC) algorithm for drug-DNA docking that accounts for the molecular flexibility of both constituents and samples the docking geometry without any prior binding-site selection. The binding of the antimalarial drug methylene blue at the DNA minor groove with a preference of binding to AT-rich over GC-rich base sequences is obtained in MC simulations in accordance with experimental data. In addition, the transition between two drug-DNA-binding modes, intercalation and minor-groove binding, has been achieved in dependence on the DNA base sequence. The reliable ab initio prediction of drug-DNA binding achieved by our new MC docking algorithm is an important step towards a realistic description of the structure and dynamics of molecular recognition in biological systems.

A Small Molecule Inhibitor of Bruton's Tyrosine Kinase Involved in B-Cell Signaling.

Protein kinases are fundamental within almost all cellular signal transduction networks. Among these, Bruton's tyrosine kinase (Btk), which belongs to the Tec family of proteins, plays an imperative part in B-cell signaling. Owing to its role, Btk has been established as an important therapeutic target for a vast range of disorders related to B-cell development and function, such as the X-linked agammaglobulinemia, various B-cell malignancies, inflammation, and autoimmune diseases. Herein, using computer-based screening of a library of 20 million small molecules, we identified a small molecule capable of directly binding the Btk kinase domain. On the basis of this hit compound, we conducted a focused structure-similarity search to explore the effect of different chemical modifications on binding toward Btk. This search identified the molecule N2,N6-bis(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-9H-purine-2,6-diamine as a potent inhibitor of Btk. The latter small molecule binds Btk with a dissociation constant of 250 nM and inhibits Btk activity both in vitro and in-cell.

D-enantiomer peptide of the TCRalpha transmembrane domain inhibits T-cell activation in vitro and in vivo.

T cell activation requires the cross-talk between the CD3-signaling complex and the T cell receptor (TCR). A synthetic peptide coding for the TCRalpha transmembrane domain (CP) binds CD3 molecules, interferes with the CD3/TCR cross-talk, and inhibits T cell activation. Intermolecular interactions are sterically constrained; accordingly no sequence-specific interactions are thought to occur between D- and L-stereoisomers. This argument was recently challenged when applied to intra-membrane protein assembly. In this paper we studied the ability of a D-stereoisomer of CP (D-CP) to inhibit T cell activation. L-CP and D-CP co-localized with the TCR in the membrane and inhibited T cell activation in a sequence-specific manner. In vivo, both L-CP and D-CP inhibited adjuvant arthritis. In molecular terms, these results suggest the occurrence of structural reorientation that facilitates native-like interactions between D-CP and CD3 within the membrane. In clinical terms, our results demonstrate that D-stereoisomers retain the therapeutic properties of their L-stereoisomers, while they benefit from an increased resistance to degradation.

Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

Specificity in transmembrane helix-helix interactions mediated by aromatic residues.

Aromatic residues have been previously shown to mediate the self-assembly of different soluble proteins through pi-pi interactions (McGaughey, G. B., Gagne, M., and Rappe, A. K. (1998) J. Biol. Chem. 273, 15458-15463). However, their role in transmembrane (TM) assembly is not yet clear. In this study, we performed statistical analysis of the frequency of occurrence of aromatic pairs in a bacterial TM data base that provided an initial indication that the appearance of a specific aromatic pattern, Aromatic-XX-Aromatic, is not coincidental, similar to the well characterized QXXS motif. The QXXS motif was previously shown to be both critical and sufficient for stabilizing TM self-assembly. Using the ToxR system, we monitored the dimerization propensities of TM domains that contain mutations of interacting residues to aromatic amino acids and demonstrated that aromatic residues can adequately stabilize self-association. Importantly, we have provided an example of a natural TM domain, the cholera toxin secretion protein EpsM, whose TM self-assembly is mediated by an aromatic motif (WXXW). This is, in fact, the first evidence that aromatic residues are involved in the dimerization of a wild type TM domain. The association mediated by aromatic residues was found to be sensitive to the TM sequence, suggesting that aromatic residue motifs can provide a general means for specificity in TM assembly. Molecular dynamics provided a structural explanation for this backbone sequence sensitivity.

A structurally altered D,L-amino acid TCRalpha transmembrane peptide interacts with the TCRalpha and inhibits T-cell activation in vitro and in an animal model.

Protein-protein interactions in the membrane are pivotal for the cellular response to receptor-sensed stimuli. Recently, it has been demonstrated that an all-d-amino acids analogue of the TCRalpha transmembrane peptide (CP) is recruited to the TCR complex and inhibits T-cell activation in vitro and in vivo, similarly to the wild-type CP peptide. Here we investigated the relative contributions of the secondary structure of CP compared to its side chains in the association of CP with the TCR. We disrupted the secondary structure of CP by replacing two positive residues, needed for the interaction of CP with the TCR complex, by their d-enantiomers (2D-CP). Structure disruption was demonstrated by CD and FTIR spectroscopy, and molecular dynamics simulation in a bilayer environment. In vitro, 2D-CP colocalized with the TCR (visualized with confocal microscopy), immunoprecipitated with TCR but not MHC I, and inhibited T-cell activation. The peptide was effective also in vivo: it inhibited adjuvant arthritis in rats and delayed type hypersensitivity in BALB/c mice. Moreover, 2D-CP manifested greater immunosuppressive activity than wild-type CP, both in vivo and in vitro, which can be attributed to the greater solubility and resistance to degradation of 2D-CP. In molecular terms, these findings suggest that, under certain conditions, protein-protein interactions within the membrane might be more dependent on side chain interactions than on a specific secondary structure. The new altered secondary structure probably determines how the Lys and the Arg are positioned with respect to each other, so they can interact with the TM domain of the receptor. In clinical terms, the increased solubility and resistance to degradation of d-stereoisomers might be exploited in the targeted inactivation of pathogenic signaling pathways such as those arising from TCR-triggered activation of T-cells in immune-mediated disorders.