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SARS-CoV and MERS-CoV Macro Domains (MDs) exhibit topological and conformational features that resemble the nsP3b macro (or “X”) domain of SARS-CoV-2. Indeed, all the three domains (SARS-CoV-2, SARS-CoV and MERS-CoV MDs) fold in a three-layer α/β/α sandwich structure, as reported by crystallographic structural investigation of SARS-CoV MD and MERS-CoV MD. These viral MDs are able to bind ADP-ribose as many other MDs from different kingdoms. They have been characterized also as de-ADP-ribosylating enzymes. For this reason, these viral macrodomains recently emerged as important drug targets since they can counteract antiviral ADP-ribosylation mediated by poly-ADP-ribose polymerase (PARPs). Even in presence of the 3D structures of SARS-CoV MD and of MERS-CoV MD, we report herein the almost complete NMR backbone (1H, 13C, 15N) of SARS-CoV MD and MERS-CoV proteins in the free and ADPr bound forms, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will help to further understanding of the atomic-level conformational dynamics of these proteins and will allow an extensive screening of small molecules as potential antiviral drugs.

hPARP14 is a human ADP-ribosyl-transferase (ART) that belongs to the macroPARPs family, together with hPARP9 and hPARP15. It contains a tandem of three macro domains (MD) while each of them has different properties. The first one, namely MD1, has not been reported to exhibit a high binding affinity for ADP-ribose (ADPr) in contrast to the following two (MD2 and MD3). All three MDs exhibit an α/β/α sandwich-like fold as reported by the deposited crystallographic structures. MD2 and MD3 recognize mono-ADP-ribosylated (MARylated) but not poly-ADP-ribosylated (PARylated) substrates and thus they allow hPARP14 to bind its targets, which can be potentially MARylated by its catalytic domain (CD). hPARP14 participates in DNA damage repair process and immune response against viruses like SARS-CoV-2, which also harbors an MD fold. Furthermore, hPARP14 like the other two macroPARPs (hPARP9 and hPARP15), is implicated in numerous types of cancer, such as B-aggressive lymphoma and sarcoma, rendering its MDs as potential important drug targets. Herein, we report the complete NMR backbone and side chain assignment (

Losartan potassium salt (LSR) is a well-known antihypertensive drug with proven beneficial effects on human health. Its formulation with the non-toxic 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) could improve its pharmacological profile. Thus, its molecular interactions are studied using a combination of Differential Scanning Calorimetry (DSC), Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD). First, its complexation is shown through Differential Scanning Calorimetry as lyophilization provided distinct thermal properties in comparison to the mixture. The complexation is further proved by utilizing the chemical shift changes in the complexation and T1 values. Furthermore, the reversible favorable complexation was shown by MD calculations. Such physical chemical properties provide evidence that this formulation must be further explored through biological experiments.

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.

Neurotensin (NT) (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) exerts a dual function as a neurotransmitter/neuromodulator in the central nervous system and as a hormone/cellular mediator in periphery. This dual function of NT establishes a connection between brain and peripheral tissues that renders this peptide a central player in energy homeostasis. Many biological actions of NT are mediated through its interaction with three types of NT receptors (NTS receptors). Despite its role in energy homeostasis, NT has a short half-life that hampers further determination of the biological actions of this peptide and its receptors in brain and periphery. The short half-life of NT is due to the proteolytic degradation of its C-terminal side by several endopeptidases. Therefore, it is important to synthesize NT analogues with resistant bonds against metabolic deactivation. Based on these findings, we herein report the synthesis of ten linear, two cyclic and two dimeric analogues of NT with modifications in its structure that improve their metabolic stability, while retaining the ability to bind to NTS receptors. Modifications at position 11 (introduction of D-Tyrosine (OEthyl) [D-Tyr(Et)] or D-1-naphtylalanine [D-1-Nal] were combined with introduction of a L-Lysine or a D-Arginine at positions 8 or 9, and 1-[2-(aminophenyl)-2-oxoethyl]-1H-pyrrole-2-carboxylic acid (AOPC) at positions 7 or 8, resulting in compounds NT4-NT21. AOPC is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetic compounds. To biologically evaluate these analogues, we determined their plasma stability and their binding affinities to type 1 NT receptor (NTS1), endogenously expressed in HT-29 cells, Among the fourteen NT analogues, compounds, NT5, NT6, and NT8, which have D-Tyr(Et) at position 11, bound to NTS1 in a dose-response manner and with relatively high affinity but still lower than that of the natural peptide. Despite their lower binding affinities compared to NT, the NT5, NT6, and NT8 exhibited a remarkably higher stability, as a result of their chemistry, which provides protection from enzymatic activity. These results will set the basis for the rational design of novel NT molecules with improved pharmacological properties and enhanced enzymatic stability.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.

Context Inhibitors of sodium-glucose cotransporters-2 have cardio- and renoprotective properties. However, the underlying mechanisms remain indeterminate. Objective To evaluate the effect of dapagliflozin on renal metabolism assessed by urine metabolome analysis in patients with type 2 diabetes. Design Prospective cohort study. Setting Outpatient diabetes clinic of a tertiary academic center. Patients Eighty patients with hemoglobin A1c > 7% on metformin monotherapy were prospectively enrolled. Intervention Fifty patients were treated with dapagliflozin for 3 months. To exclude that the changes observed in urine metabolome were merely the result of the improvement in glycemia, 30 patients treated with insulin degludec were used for comparison. Main Outcome Measure Changes in urine metabolic profile before and after the administration of dapagliflozin and insulin degludec were assessed by proton-nuclear magnetic resonance spectroscopy. Results In multivariate analysis urine metabolome was significantly altered by dapagliflozin (R2X = 0.819, R2Y = 0.627, Q2Y = 0.362, and coefficient of variation analysis of variance, P Conclusions Dapagliflozin significantly affects urine metabolome in patients with type 2 diabetes in a glucose lowering-independent way. Most of the observed changes can be considered beneficial and may contribute to the renoprotective properties of dapagliflozin.

La is an abundant phosphoprotein that protects polymerase III transcripts from 3'-5' exonucleolytic degradation and facilitates their folding. Consisting of the evolutionary conserved La motif (LAM) and two consecutive RNA Recognition Motifs (RRMs), La was also found to bind additional RNA transcripts or RNA domains like internal ribosome entry site (IRES), through sequence-independent binding modes which are poorly understood. Although it has been reported overexpressed in certain cancer types and depletion of its expression sensitizes cancer cells to certain chemotherapeutic agents, its role in cancer remains essentially uncharacterized. Herein, we study the effects of La overexpression in A549 lung adenocarcinoma cells, which leads to increased cell proliferation and motility. Expression profiling of several transcription and translation factors indicated that La overexpression leads to downregulation of global translation through hypophosphorylation of 4E-BPs and upregulation of IRES-mediated translation. Moreover, analysis of La localization after nutrition deprivation of the transfected cells showed a normal distribution in the nucleus and nucleoli. Although the RNA binding capacity of La has been primarily linked to the synergy between the conserved LAM and RRM1 domains which act as a module, we show that recombinant stand-alone LAM can specifically bind a pre-tRNA ligand, based on binding experiments combined with NMR analysis. We propose that LAM RNA binding properties could support the expanding and diverse RNA ligand repertoire of La, thus promoting its modulatory role, both under normal and pathogenic conditions like cancer.

Organotin(IV) derivatives of cholic acid (CAH) with the formulae R3Sn(CA) (R = Ph- (1), n-Bu- (2)) and R2Sn(CA)2 (R = Ph- (3), n-Bu- (4) and Me- (5)) were synthesized. The compounds were characterized in solid state by melting point, FT-IR, 119Sn Mossbauer, X-ray fluorescence (XRF) spectroscopy and in solution by 1H NMR, UV–Vis spectral data and by Electrospray Ionisation Mass spectrometry (ESI-MS), High Resolution Mass spectrometry (HRMS), and atomic absorption analysis. The in vitro bioactivity of 1–5 against human breast adenocarcinoma cancer cells MCF-7 (positive to hormone receptors) and MDA-MB-231 (negative to hormone receptors) reveal that triorganotin derivatives 1–2 exhibit significantly stronger activity than the corresponding diorganotin ones. Compound 5 is inactive against both cell lines at the concentrations tested. Triorganotins 1–2 inhibit selectively MCF-7 than MDA-MB-231 cells, suggesting hormone mimetic behavior of them. Organotins 1–4 inhibit both cancerous cell lines, stronger than cisplatin which rise up to 55-fold against MCF-7 and 170-fold against MDA-MB-231. The in vitro toxicity of 1–4 was evaluated on normal human fetal lung fibroblast cells (MRC-5), while their genotoxicity in vitro by micronucleus assay (MN). Moreover, the in vivo toxicity of 1–4 was tested by Artemia salina assay and their in vivo genotoxicity with Allium cepa test. The mechanism of action of 1–4 against MCF-7 was clarified in vitro by the means of cell morphology studies, cell cycle arrest, Acridine Orange/Ethidium Bromide (AO/EB) Staining, mitochondrial membrane permeabilization test and by their binding affinity toward the calf thymus (CT) DNA.

A major challenge from the moment a child is delivered is the adaptation to the extrauterine life, where rapid metabolic changes take place. The study of these changes during the first days of human life may assist in the understanding of the metabolic processes that occur at this critical period, which is likely to provide significant clinical insights. To date, metabolomics has become a powerful field, ideal for the monitoring of such dynamic variations, since it offers the possibility to identify alterations in metabolic profiles, even on daily basis. The study included 253 healthy newborns (GA 35 to 40 weeks) from the region of Western Greece. Urine samples were collected immediately after birth and at the third day of life. NMR-based metabolomics was used to compare the metabolic urinary profiles of newborns from the first and third day of their life, assessing the impact of six perinatal factors; delivery mode, prematurity, maternal smoking, gender, nutrition and neonatal jaundice. Analysis of urine metabolic fingerprint from the first and third day of life, coupled with multivariate statistics, provides insights into the details of early life metabolic profile differentiation. Αt the third day of life metabolic adaptations are evident, as many differences were noted in urine of healthy neonates within the first 72 h postpartum. Trends in differentiation of metabolites levels between the two groups, late preterm and term newborns, have been also observed. Newborn’s urine metabolic profiles confirmed the rapid changes in their metabolism after birth. Further, ongoing research will enable us to develop one reference model of urinary metabolomics in healthy newborns during the period of adaptation to the extra-uterine life.

Blood is one of the most used biofluids in metabolomics studies, and the serum and plasma fractions are routinely used as a proxy for blood itself. Here we investigated the association networks of an array of 29 metabolites identified and quantified via NMR in the plasma and serum samples of two cohorts of ∼1000 healthy blood donors each. A second study of 377 individuals was used to extract plasma and serum samples from the same individual on which a set of 122 metabolites were detected and quantified using FIA-MS/MS. Four different inference algorithms (ARANCE, CLR, CORR, and PCLRC) were used to obtain consensus networks. The plasma and serum networks obtained from different studies showed different topological properties with the serum network being more connected than the plasma network. On a global level, metabolite association networks from plasma and serum fractions obtained from the same blood sample of healthy people show similar topologies, and at a local level, some differences arise like in the case of amino acids.

Surface active agents are characterized for their capacity to adsorb to fluid and solid-water interfaces. They can be classified as surfactants and emulsifiers based on their molecular weight (MW) and properties. Over the years, the chemical surfactant industry has been rapidly increasing to meet consumer demands. Consequently, such a boost has led to the search for more sustainable and biodegradable alternatives, as chemical surfactants are non-biodegradable, thus causing an adverse effect on the environment. To these ends, many microbial and/or marine-derived molecules have been shown to possess various biological properties that could allow manufacturers to make additional health-promoting claims for their products. Our aim, in this review article, is to provide up to date information of critical health-promoting properties of these molecules and their use in blue-based biotechnology (i.e., biotechnology using aquatic organisms) with a focus on food, cosmetic and pharmaceutical/biomedical applications.

Background: The intrinsically disordered, amyloidogenic protein Tau associates with diverse classes of molecules, including proteins, nucleic acids, and lipids. Mounting evidence suggests that fatty acid molecules could play a role in the dysfunction of this protein, however, their interaction with Tau remains poorly characterized. Methods: In a bid to elucidate the association of Tau with unsaturated fatty acids at the sub-molecular level, we carried out a variety of solution NMR experiments in combination with circular dichroism and fluorescence measurements. Our study shows that Tau4RD, the highly basic four-repeat domain of Tau, associates strongly with arachidonic and oleic acid assemblies in a high lipid/protein ratio, perturbing their supramolecular states and itself undergoing time-dependent structural adaptation. The structural signatures of Tau4RD/fatty acid aggregates appear similar for arachidonic acid and oleic acid, however, they are distinct from those of another prototypical intrinsically disordered protein, &alpha, synuclein, when bound to these lipids, revealing protein-specific conformational adaptations. Both fatty acid molecules are found to invariably promote the self-aggregation of Tau4RD and of &alpha, synuclein. Conclusions: This study describes the reciprocal influence that Tau4RD and fatty acids exert on their conformational states, contributing to our understanding of fundamental aspects of Tau/lipid co-assembly.

The corticotropin-releasing factor (CRF) and its CRF1 receptor (CRF(1)R) play a central role in the maintenance of homeostasis. Malfunctioning of the CRF/CRF(1)R unit is associated with several disorders, such as anxiety and depression. Non-peptide CRF(1)R-selective antagonists have been shown to exert anxiolytic and antidepressant effects on experimental animals. However, none of them is in clinical use today because of several side effects, thus demonstrating the need for the development of other more suitable CRF(1)R antagonists. In an effort to develop novel CRF(1)R antagonists we designed, synthesized and chemically characterized two tripeptide analogues of CRF, namely (R)-LMI and (S)-LMI, having their Leu either in R (or D) or in S (or L) configuration, respectively. Their design was based on the crystal structure of the N-extracellular domain (N-domain) of CRF(1)R/CRF complex, using a relevant array of computational methods. Experimental evaluation of the stability of synthetic peptides in human plasma has revealed that (R)-LMI is proteolytically more stable than (S)-LMI. Based on this finding, (R)-LMI was selected for pharmacological characterization. We have found that (R)-LMI is a CRF antagonist, inhibiting (1) the CRF-stimulated accumulation of cAMP in HEK 293 cells expressing the CRF(1)R, (2) the production of interleukins by adipocytes and (3) the proliferation rate of RAW 264.7 cells. (R)-LMI likely blocked agonist actions by interacting with the N-domain of CRF(1)R as suggested by data using a constitutively active chimera of CRF(1)R. We propose that (R)-LMI can be used as an optimal lead compound in the rational design of novel CRF antagonists.

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical “macro” (ββαββαβαβα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD–ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP‐r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure–function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions., Graphical abstract Unlabelled Image, Highlights • First conformational and dynamics study of a previously unexplored Alphavirus macro domain (Mayaro virus macro domain, or MAYV MD) • MAYV MD selectivity and binding of ADP-ribose revealed by NMR spectroscopy and ITC • Identification of critical residues for the ADP-ribose binding • MAYV MD preference for binding of RNAs rich in adenine observed by NMR spectroscopy • Binding studies provide new insights for the design of novel ligands for MAYV and other Alphaviruses MDs.

Graphical abstract, Highlights • ADPr’s binding triggers conformational changes to the whole VEEV macro domain. • High flexibility of the loops β5-α3 and α3-β6 assist the ADPr’s binding. • Loops around ADPr site undergo a transition pathway between apo and complex state., Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops β5-α3 and α3-β6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated “dark” conformational state.

The growth hormone-releasing hormone receptor (GHRHR) is a member of the class B GPCR subfamily. GHRH, a 44-residue neuropeptide produced in the hypothalamus, regulates the secretion of growth hormone through its binding to GHRHR. It has recently been associated with several types of cancer such as prostate, breast, pancreatic and ovarian cancer. Family B GPCR peptides bind in a two-step model, where first the C-terminal region of the peptide interacts with the extracellular domain (ECD) of the receptor and subsequently, the N-terminal interacts with the seven transmembrane domain (TMD), resulting in activation. Structural information on family B GPCRs is limited; therefore, the use of computational methods may assist their efficient targeting towards new therapeutics. Here, we have utilized several computational tools, such as homology modelling, docking, large-scale molecular dynamics and principal component analysis (PCA), in order to: (a) gain information on the dynamic properties of the receptor domains and (b) propose a structural model for the interactions between GHRH and the ECD and TMD regions of GHRHR respectively. We conclude that PCA analysis can be used for studying such relative movements in family B GPCRs and provide a structural model, which may assist in the design of highly anticipated non-peptide antagonists against GHRHR.

The soluble guanylate cyclase (sGC) is the physiological sensor for nitric oxide and alterations of its function are actively implicated in a wide variety of pathophysiological conditions. Intense research efforts over the past 20 years have provided significant information on its regulation, culminating in the rational development of approved drugs or investigational lead molecules, which target and interact with sGC through novel mechanisms. However, there are numerous questions that remain unanswered. Ongoing investigations, with the critical aid of structural chemistry studies, try to further elucidate the enzyme’s structural characteristics that define the association of “stimulators” or “activators” of sGC in the presence or absence of the heme moiety, respectively, as well as the precise conformational attributes that will allow the design of more innovative and effective drugs. This review relates the progress achieved, particularly in the past 10 years, in understanding the function of this enzyme, and focusses on a) the rationale and results of its therapeutic targeting in disease situations, depending on the state of enzyme (oxidized or not, heme-carrying or not) and b) the most recent structural studies, which should permit improved design of future therapeutic molecules that aim to directly upregulate the activity of sGC.

A member of the ribonuclease A superfamily, human angiogenin (hAng) is a potent angiogenic factor. Heteronuclear NMR spectroscopy combined with induced-fit docking revealed a dual binding mode for the most antiangiogenic compound of a series of ribofuranosyl pyrimidine nucleosides that strongly inhibit hAng's angiogenic activity in vivo. While modeling suggests the potential for simultaneous binding of the inhibitors at the active and cell-binding sites, NMR studies indicate greater affinity for the cell-binding site than for the active site. Additionally, molecular dynamics simulations at 100 ns confirmed the stability of binding at the cell-binding site with the predicted protein-ligand interactions, in excellent agreement with the NMR data. This is the first time that a nucleoside inhibitor is reported to completely inhibit the angiogenic activity of hAng in vivo by exerting dual inhibitory activity on hAng, blocking both the entrance of hAng into the cell and its ribonucleolytic activity.

Here, we report the high yield expression and preliminary structural analysis via solution hetero-nuclear NMR spectroscopy of the recombinant Met-1 human Angiogenin. The analysis reveals a well folded as well as, a monomeric polypeptide. Τhe sequence-specific assignment of its (1)H, (15)N and (13)C resonances at high percentage was obtained. Also, using TALOS+ its secondary structure elements were determined.

Arkadia (Rnf111), an E3 Ubiquitin (Ub) ligase, amplifies TGF-β signaling responses by targeting for degradation of the negative regulators Smad6/7 and the SnoN/Ski transcriptional repressors when they block the TGF-β effectors Smad2/3. The E3 ligase activity of Arkadia depends on its C-terminal RING-H2 domain that constitutes the docking site for the E2 Ub-conjugating enzyme carrying the activated Ub. We determined the nuclear magnetic resonance solution structure of Arkadia's RING-H2 domain and revealed a (β)ββα fold, fully consistent with the expected "cross-brace" mode of Zn(II)-ligation. In addition, the interaction of the Arkadia RING-H2 domain with its E2 partner enzyme (UbcH5b) was examined through chemical shift perturbation. Proteins 2012. © 2012 Wiley Periodicals, Inc.

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both ανβ3 integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN112–136) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN112–136 inhibited binding of PTN to ανβ3 integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β3. Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing ανβ3-selective antibody. PTN112–136 did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β3 Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN112–136 may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN112–136 was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123–132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN112–136 induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to ανβ3 integrin. J. Cell. Biochem. 112: 1532–1543, 2011. © 2011 Wiley-Liss, Inc.

Combined blockade of the renin-angiotensin-aldosterone system (RAAS) is an attractive therapeutic strategy for the treatment of cardiovascular diseases. Vasopeptidase inhibitors are a group of compounds capable of inhibiting more than one enzyme, which leads to potentiation of natriuretic peptide actions and suppression of the RAAS. In this study, molecular modeling has been used to elucidate key structural features that govern the binding and/or selectivity of a single compound toward the zinc catalytic sites of the N- and C-domains of the angiotensin-converting enzyme (ACE) and the neutral endopeptidase (NEP). Eleven dual inhibitors were categorized in three classes, according to their zinc binding groups. Analysis of their docked conformers revealed the molecular environment of the catalytic sites and the specific interactions between the inhibitors and amino acid residues that are important for selectivity and cooperativity. In addition, inhibitors were predicted to bind to the C-domain of the ACE with greater affinity than the N-domain, with an average difference in the free energy of binding approximately 2-3 kcal mol(-1). Residues that were identified to actively participate in the binding and stabilizing of the enzyme-inhibitor complexes were analyzed in a consensus way for both the ACE and the NEP. These atomic-level insights into enzyme-ligand binding can be used to drive new structure-based drug design processes in the quest for more selective and effective vasopeptidase inhibitors.

Anthrax Lethal Factor (LF) is a zinc-dependent metalloprotease that together with the protective antigen constitute the anthrax lethal toxin, the most prominent virulence factor of the disease anthrax. This review summarizes the current knowledge on anthrax toxicity and defense in relation to LF. Particular emphasis is placed on the structural aspects of LF, the properties of its substrates and the achievements in the design of low molecular weight inhibitors of the catalytic activity of the metalloenzyme.

Properdin is a plasma glycoprotein and the only known naturally occurring positive regulator of the complement system, stabilizing the alternative pathway convertase (C3bBb). In order to elucidate the molecular evolution of properdin factor P (pfc), here we report the cloning and characterization of three gene isoforms of properdin in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout properdins pfc1, pfc2 and pfc3 (447, 449 and 447 amino acids, respectively) share 78-90% identity to each other, showing the highest identity score (47%) with their zebrafish ortholog protein. The overall identity with human, mouse and xenopus properdin polypeptides is 44%, 42% and 45%, respectively. The 'domain' architecture of trout properdins resembles that of the mammalian counterpart proteins, composed of six thrombospondin repeat type 1 domains (TSR-1-TSR-6). TSR domains of the three trout properdin isoforms seem to adopt the folding pattern of human thrombospondin 1 TSP-1 domains, where each TSP-1 domain forms an antiparallel three-stranded structure that consists of alternative stacked layers of Trp and Arg residues from respective strands capped by disulfide bonds on each end. The trout pfc2 and pfc3 genes are arranged in nine and ten exons, respectively, which span approximately 3.5kb of the genome. In contrast to the expression profile of the properdin gene in mammals, liver is the main source of the trout properdin mRNA transcripts. In a phylogenetic analysis, trout pfc1, pfc2 and pfc3 genes are clustered with their orthologs from other teleost species. This is the first report of three separate genes coding for properdin factor P in a vertebrate species.

Human angiotensin-I converting enzyme (ACE) is a central component of the renin-angiotensin system and a major target for cardiovascular therapies. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. On the basis of the recently determined crystal structures of both ACE domains, we have studied their complexes with gonadotropin-releasing hormone (GnRH), which is cleaved releasing both the protected NH2- and COOH-terminal tripeptides. This is the first molecular modeling study of an ACE-peptide substrate complex that examines the structural basis of ACE's endopeptidase activity and offers novel insights into subsites that are distant from the obligatory binding site and were not identified in the crystal structures. Our data indicate that a bridging interaction between Arg500 of the N-domain and Arg8 of GnRH that involves a buried chloride ion may account for its role in the specificity of the N-domain for endoproteolytic cleavage of the substrate at the NH2-terminus in vitro. In support of this, the protected NH2-terminal dipeptide of GnRH exhibits stronger interactions than the protected COOH-terminal dipeptide with the N-domain of ACE. Further comparison of the models of ACE-substrate complexes promotes our understanding of how the two domains differ in their function and specificity and provides an extension of the pharmacophore model used for structure-based drug design up to the S7 subsite of the enzyme.

Angiotensin-I Converting Enzyme (ACE) is a Zinc Metallopeptidase of which the three-dimensional structure was unknown until recently, when the X-ray structure of testis isoform (C-terminal domain of somatic) was determined. ACE plays an important role in the regulation of blood pressure due to its action in the frame of the Renin-Angiotensin System. Efforts for the specific inhibition of the catalytic function of this enzyme have been made on the basis of the X-ray structures of other enzymes with analogous efficacy in the hydrolytic cleavage of peptide substrate terminal fragments. Angiotensin-I Converting Enzyme bears the sequence and topology characteristics of the well-known gluzincins, a sub-family of zincins metallopeptidases and these similarities are exploited in order to reveal common structural elements among these enzymes. 3D homology models are also built using the X-ray structure of Thermolysin as template and peptide models that represent the amino acid sequence of the ACE's two catalytic, zinc-containing sites are designed and synthesized. Conformational analysis of the zinc-free and zinc-bound peptides through high resolution 1H NMR Spectroscopy provides new insights into the solution structure of ACE catalytic centers. Structural properties of these peptides could provide valuable information towards the design and preparation of new potent ACE inhibitors.

The La protein (Lupus antigen), a key mediator during biogenesis of RNA polymerase III transcripts, contains a characteristic La motif and one or two RNA recognition motif (RRM) domains, depending on the organism of origin. The RRM1 domain is conserved in higher eukaryotes and located in the N-terminal region, whereas the C-terminal RRM2 domain is absent in most lower eukaryotes and its specific role remains, so far, uncharacterized. Here, we present the backbone and side-chain assignment of the (1)H, (13)C and (15)N resonances of RRM2 of La protein from Dictyostelium discoideum. Interestingly, the La protein in this lower eukaryote, exhibits high homology to its human counterpart. Moreover, it contains two RRM domains, instead of one, raising questions on its evolutionary origin and the putative role of RRM2 in vivo. We also provide its secondary structure as predicted by the TALOS+ online tool.

Anthrax lethal factor (LF) is a zinc-metalloprotease that together with the protective antigen constitutes anthrax lethal toxin, which is the most prominent virulence factor of the anthrax disease. The solution nuclear magnetic resonance and in silico conformational dynamics of the 105 C-terminal residues of the LF catalytic core domain in its apo form are described here. The polypeptide adopts a compact structure even in the absence of the Zn(2+) cofactor, while the 40 N-terminal residues comprising the metal ligands and residues that participate in substrate and inhibitor recognition exhibit more flexibility than the C-terminal region.

We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H???S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H???S are cystathionine ?? synthase (CBS) and cystathionine ?? lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H???S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: ??-Cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC?????? 14 ± 0.2 ??M vs. 40 ± 8 ??M respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC?????? 1.1 ± 0.1?????M); the IC?????? for AOAA for inhibiting CBS was 8.5 ± 0.7 ??M. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H???S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC?????? for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Based on the crystal structure of the extracellular domain (ECD) of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to α-bungarotoxin (α-Btx) we have generated in silico models of the human nAChR α1 bound to α-Btx and α-cobratoxin (α-Cbtx), both in the presence and in the absence of the N-linked carbohydrate chain. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. An interesting observation during the course of the MD simulations is the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain, mediated through a shift in the position of the sugars towards the bound toxin. Critical protein-sugar interactions implicate residues Ser187 and Trp184 of nAChR and Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Analysis of the predicted residue-specific intermolecular interactions is intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism.

Amyloid beta peptide (Abeta) aggregation leads to the senile plaque formation, a process that is strongly influenced by oxidative stress and is considered as the molecular basis of various neurodegenerative diseases, such as Alzheimer's disease (AD). Endogenous antioxidants or dietary derived compounds may down-regulate this process. In this study, the interaction of two antioxidants, oleuropein (OE) and melatonin (M), with Abeta is monitored through nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The concerted application of these two analytical techniques provides new experimental evidence and residue-specific insights into the interacting Abeta peptide amino acids that are implicated in this process. Both antioxidant compounds interact in a similar way with the peptide and cause chemical shift variations. The most pronounced resonance changes have been observed for the 1H-15N signals of N-terminal region and Leu17-Phe20 residues, as monitored by NMR titration studies.

Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

Human ACE is a central component of the renin-angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE.

The RING (Really Interesting New Gene) family is the largest type of E3 ubiquitin ligases. RING finger domains bind two zinc ions in a unique "cross-brace" arrangement through a defined motif of cysteine and histidine residues. This arrangement endows the RING domain with a globular conformation, characterized by a central alpha-helix and variable-length loops separated by several small beta-strands. RING E3 ubiquitin ligases, play an essential role in the regulation of many biologic processes and defects in some of them are involved in cancer development. Furthermore, some RING E3 ligases are frequently overexpressed in human cancers. Today, RING ligases represent potentially molecular targets for disease intervention and could act as prognostic biomarkers. Targeting specific RING E3 ligases could lead to the development of a novel class of anticancer drugs. However RING fingers exhibit remarkable variations in their sequence and their topology characteristics. Structure determination of new RING finger domain is in the core of the design of new pharmaceuticals and what is presented in this article is a thorough review of achievements on the NMR or Xray structure determinations. Protein preparation protocols along with analysis of the structural features of known RING finger are also presented.

Angiotensin-converting enzyme (ACE) is a key molecule of the renin-angiotensin-aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala(959) to Ser(1066) region (ACE_C) that corresponds to the C-catalytic domain of human somatic angiotensin-I-converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1-trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE(959-1066) protein fragment in order to study its structure in solution by NMR spectroscopy.

Myelin basic protein peptide 83-99 (MBP83-99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP(83-99) has been performed using 2D (1)H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined alpha-helix spanning residues Val(87)-Phe(90) is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.

We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361 to Gly468, ACE-N) of human somatic angiotensin-I converting enzyme in Escherichia coli. This construct represents the N-catalytic domain including the two binding motifs and the 23 amino acid spacers as well as some amino acid residues before and after the motifs that might help in correct conformation. The overexpressed protein was exclusively localized to insoluble inclusion bodies. Inclusion bodies were solubilized in an 8-M urea buffer. Purification was carried out by differential centrifugation and gel filtration chromatography under denaturing conditions. About 12 mg of ACE-N peptide per liter of bacterial culture was obtained. The integrity of recombinant protein domain was confirmed by ESI/MS. Structural analysis using CD spectroscopy has shown that, in the presence of TFE, the ACE-N protein fragment has taken a conformation, which is consistent with the one found in testis ACE by X-ray crystallography. This purification procedure enables the production of an isotopically labeled protein fragment for structural studying in solution by NMR spectroscopy. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.

Infection of target host cells by the human immunodeficiency virus-1 (HIV-1) is a multi-step process involving a series of conformational changes in the viral gp120 and gp41 proteins. Gp120 binding to the main cell receptor, CD4, on the surface of cells expressing this molecule, and interaction with the cell chemokine receptors CCR5 and CXCR4, are among the key events for HIV-1 infection. These steps are crucial for the virus and offer potential therapeutic targets. For this reason, understanding the structure and the physicochemical characteristics of the gp120 in relation to these interactions has drawn much attention. This review article focuses on the biologically important V3 region of the gp120 and summarizes the functional role, the sequence variation and the conformational features of V3 peptides, which are important for co-receptor selectivity, specificity and interaction. Synthetic V3 peptides have been extensively studied by NMR spectroscopy and X-ray crystallography, in solution or in solid state, in their free or bound form, and valuable information was generated with the aim to be exploited in the design of new, effective inhibitors of HIV-1 infection. The features of the potential gp120 interacting sites on the two chemokine co-receptors, CCR5 and CXCR4, are also discussed, and co-receptor blocking molecules under clinical trial are also reported.

The solution NMR conformational properties of two angiotensin converting enzyme (ACE) Zn catalytic-site 36-residue peptides, with the general sequence HEMGHX23EAIGDX3, synthesized through solid-phase 9-flourenylmethyoxycarbonyl (Fmoc) chemistry, is reported. The 1H resonance assignment of Zn-bound peptides is presented and the characteristic features of the NMR solution models of the two ACE Zn(II)-bound peptides are reported. The solid-state and solution structures of the ACE C-domain catalytic site are compared while biologically important structural similarities and differences of the N- and C-terminal catalytic sites are discussed. Additionally, the structural features of the ACE substrate, the angiotensin I (AI) decapeptide, are studied using NMR spectroscopy, in order to set the structural basis for the ACE–substrate interaction in solution. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two sequence homologous protein domains (ACE_N and ACE_C) possessing several biochemical features that differentiate the two active sites (i.e. chloride ion activation). Based on the recently solved X-ray structure of testis angiotensin-converting enzyme (tACE), the 3D structure of ACE_N was modeled. Electrostatic potential calculations reveal that the ACE_N binding groove is significantly more positively charged than the ACE_C, which provides a first rationalization for their functional discrimination. The chloride ion pore for Cl2 (one of the two chloride ions revealed in the X-ray structure of tACE) that connects the external solution with the inner part of the protein was identified on the basis of an extended network of water molecules. Comparison of ACE_C with the X-ray structure of the prokaryotic ClC Cl(-) channel from Salmonella enterica serovar typhimurium demonstrates a common molecular basis of anion selectivity. The critical role for Cl2 as an ionic switch is emphasized. Sequence and structural comparison between ACE_N and ACE_C and of other proteins of the gluzincin family highlights key residues that could be responsible for the peptide hydrolysis mechanism. Currently available mutational and substrate hydrolysis data for both domains are evaluated and are consistent with the predicted model.

We report the design and synthesis through solid phase 9-flourenylmethoxycarbonyl (Fmoc) chemistry of the two angiotensin-I converting enzyme active sites possessing the general sequence HEMGHX23EAIGDX3. Their zinc-binding properties were monitored in solution through high-resolution 1H-NMR. The obtained data were analyzed in terms of chemical shift differences. The results indicate that zinc binds to the HEMGH and the EAIGD characteristic motifs, and suggest possible coordination modes of zinc in the native enzyme. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 244–252, 2003

A new human/rat CRH analogue has been synthesized using the Fmoc/tBu solid-phase synthetic protocol. The sequence of the new peptide differs from the original in two positions, 12 and 15, at which the native amino acids l-phenylalanine 12 and l-leucine 15 have been replaced by the nonprotein amino acids d-phenylalanine and alpha-aminoisobutyric acid (Aib), respectively. The high resolution three-dimensional solution structure of [d-Phe12, Aib15]CRH has been determined by 688 distance constraints (656 meaningful NOE and 32 H-bonds distance limits) and 21 angle constraints. A family of 40 energy-minimized conformers was obtained with average rmsd of 0.39 +/- 0.16 A and 0.99 +/- 0.13 A for backbone and heavy atoms, respectively, and distance penalty functions of 0.42 +/- 0.03 A2. The NMR data acquired in a solvent system of water/trifluoroethanol (34%/66%, v/v) revealed that this 41-polypeptide adopts an almost linear helical structure in solution with helical content which reaches an 84% of the residues. Structural analysis confirmed the existence of two helical peptide fragments. The first was comprised of residues Ile6-Arg16 and the second of residues Glu20-Ile40, forming an angle of 34.2 degrees. The structural differences with respect to the native peptide have been identified in the region d-Phe12-Glu20 where double substitution at positions 12 and 15 seems to perturb the elements of the native 35-residue helix. These structural rearrangements promote non-native intramolecular interactions in the region of the molecule between either the hydrophobic side-chains of d-Phe12, Aib15 and Leu18, or the charged groups of the residue pairs Arg16-Glu20 and His13-Glu17 being responsible for changes in hormonal functionality. This CRH analogue currently exhibits lack of any activity.

12 pags., 4 figs., 3 tabs., 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., Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEAL