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As a cellular bile acid sensor, farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) participate in maintaining bile acid, lipid, and glucose homeostasis. To date, several selective and dual agonists have been developed as promising pharmacological approach to metabolic disorders, with most of them possessing an acidic conjugable function that might compromise their pharmacokinetic distribution. Here, guided by docking calculations, nonacidic 6-ethyl cholane derivatives have been prepared. In vitro pharmacological characterization resulted in the identification of bile acid receptor modulators with improved pharmacokinetic properties.

The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRβ and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.

Bile acids (BAs) are experiencing a new life. Next to their ancestral roles in lipid digestion and solubilization, BAs are today recognized signaling molecules involved in many physiological functions. These signaling pathways involve the activation of metabolic nuclear receptors, mainly the BA sensor FXR, and the dedicated membrane G protein-coupled receptor, GPBAR1 (TGR5). As a consequence, the discovery of GPBAR1/FXR selective or dual modulators represents an important answer to the urgent demand of new pharmacological opportunity for several human diseases including dyslipidemia, cholestasis, nonalcoholic steatohepatitis, Type 2 diabetes and inflammation. Targeted oriented discovery of natural compounds and medicinal chemistry manipulation have allowed the development of promising drug candidates.

Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders.

Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

In recent years many sterols with unusual structures and promising biological profiles have been identified from marine sources. Here we report the isolation of a series of 24-alkylated-hydroxysteroids from the soft coral Sinularia kavarattiensis, acting as pregnane X receptor (PXR) modulators. Starting from this scaffold a number of derivatives were prepared and evaluated for their ability to activate the PXR by assessing transactivation and quantifying gene expression. Our study reveals that ergost-5-en-3β-ol (4) induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target gene CYP3A4. To shed light on the molecular basis of the interaction between these ligands and PXR, we investigated, through docking simulations, the binding mechanism of the most potent compound of the series, 4, to the PXR. Our findings provide useful functional and structural information to guide further investigations and drug design.

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity.

NMDA receptors are glutamate-gated ion channels (iGluRs) that are involved in several important physiological functions such as neuronal development, synaptic plasticity, learning, and memory. Among iGluRs, NMDA receptors have been perhaps the most actively investigated for their role in chronic neurodegeneration such as Alzheimer's, Parkinson's, and Huntington's diseases. Recent studies have shown that the NTD of subunit NR2B modulates ion channel gating through the binding of allosteric modulators such as the prototypical compound ifenprodil. In the present paper, the construction of a three-dimensional model for the NR2B modulatory domain is described and docking calculations allow, for the first time, definition of the ifenprodil binding pose at an atomic level and fully explain all the available structure-activity relationships. Moreover, in an attempt to add further insight into the ifenprodil mechanism of action, as it is not completely clear if it binds and stabilizes an open or a closed conformation of the NR2B modulatory domain, a matter, which is fundamental for the rational design of NMDA antagonists, MD simulations followed by an MM-PBSA analysis were performed. These calculations reveal that the closed conformation of the R1-R2 domain, rather than the open, constitutes the high affinity binding site for ifenprodil and that a profound stabilization of the closed conformation upon ifenprodil binding occurs. Thus, for a rational design and/or for virtual screening experiments, the closed conformation of the R1-R2 domain should be taken into account and our 3D model can provide valuable hints for the design of NR2B-selective antagonists. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

We are grateful to Prof. Bernard Knoops and Andre Clippe from University of Louvain-la-Neuve, Belgium, for the generous gifts of biological materials to express the human peroxiredoxin in Escherichia coli. We thank Acellera Company for providing of a complementary 6 month ACEMD multi-GPU software licence and for the generous gift of a Nvidia Tesla K20 supercomputing unit; International audience; One of the intrinsic properties of proteins is their capacity to interact selectively with other molecules in their environment, inducing many chemical equilibria each differentiated by the mutual affinities of the components. A comprehensive understanding of these molecular binding processes at atomistic resolution requires formally the complete description of the system dynamics and statistics at the relevant time scales. While solution NMR observables are averaged over different time scales, from picosecond to second, recent new molecular dynamics protocols accelerated considerably the simulation time of realistic model systems. Based on known ligands recently discovered either by crystallography or NMR for the human peroxiredoxin 5, their affinities were for the first time accurately evaluated at atomistic resolution comparing absolute binding free-energy estimated by funnel-metadynamics simulations and solution NMR experiments. In particular, free-energy calculations are demonstrated to discriminate two closely related ligands as pyrocatechol and 4-methylpyrocathecol separated just by 1 kcal/mol in aqueous solution. The results provide a new experimental and theoretical basis for the estimation of ligand-protein affinities.

Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

Glycoproteins are often recognized as not-self molecules by antibodies triggering the onset of severe autoimmune diseases such as Multiple Sclerosis (MS). Thus, the development of antigen-mimicking biomarkers represents an attractive strategy for an early diagnosis of the disease. An example is the synthetic glycopeptide CSF114(Glc), which was designed and tested as MS biomarker and whose clinical application was limited by its reduced ability to detect autoantibodies in MS patients. In the attempt to improve the efficacy of CSF114(Glc), we have characterized all the events leading to the final binding of the biomarker to the autoantibody using atomistic simulations, ESR and NMR experiments. The glycosydic moiety plays a primary role in the whole process. In particular, in an environment mimicking that used in the clinical tests the glycopeptide assumes a α-helix structure that is functional for the interaction with the antibody. In this conformation CSF114(Glc) binds the monoclonal antibody mAb8-18C5 similarly to the myelin oligodendrocyte glycoprotein MOG, which is a known MS auto-antigen, thus explaining its diagnostic activity. Our study offers new molecular bases to design more effective biomarkers and provides a most valid protocol to investigate other systems where the environment effect is determinant for the biological activity.

Pregnane-X-receptor (PXR) is a member of nuclear receptors superfamily that activates gene transcription by binding to responsive elements in the promoter of target genes. PXR is a master gene orchestrating the expression/activity of genes involved in the metabolism of endobiotics including bilirubin, bile acids, glucose and lipid. In addition PXR oversights the metabolism of the large majority of xenobiotics including a large amount of prescribing drugs. Thus, developing PXR ligands represents a great opportunity for a therapeutic intervention on human diseases including diabetes, obesity, dyslipidemias and liver disorders. To this end, natural compounds represent an arsenal of new chemical scaffolds useful for the identification of novel PXR ligands. Here, we report a series of 4-methylenesteroid derivatives isolated from Theonella marine sponges as novel PXR modulators. In addition, combining medicinal chemistry, pharmacological experiments and computational studies, we have investigated the effects of different modifications on ring A and on the side chain of 4-methylenesteroid derivatives toward PXR modulation. This study provides the molecular bases of ligand/PXR interaction useful for designing novel PXR modulators.

Specific guanine-rich regions in human genome can form higher-order DNA structures called G-quadruplexes, which regulate many relevant biological processes. For instance, the formation of G-quadruplex at telomeres can alter cellular functions, inducing apoptosis. Thus, developing small molecules that are able to bind and sta- bilize the telomeric G-quadruplexes represents an attractive strategy for antitumor therapy. An example is 3-(benzo[d]thiazol-2-yl)-7-hydroxy-8-((4- (2-hydroxyethyl)piperazin-1-yl)methyl)-2H-chromen- 2-one (compound 1), recently identified as potent ligand of the G-quadruplex [d(TGGGGT)]4 with promising in vitro antitumor activity. The experi- mental observations are suggestive of a complex binding mechanism that, despite efforts, has defied full characterization. Here, we provide through metadynamics simulations a comprehensive un- derstanding of the binding mechanism of 1 to the G-quadruplex [d(TGGGGT)]4 . In our calculations, the ligand explores all the available binding sites on the DNA structure and the free-energy landscape of the whole binding process is computed. We have thus disclosed a peculiar hopping binding mechanism whereas 1 is able to bind both to the groove and to the 3??? end of the G-quadruplex. Our results fully explain the available experimental data, rendering our approach of great value for further ligand/DNA studies.

During the chromatographic purification of organic extracts obtained from Plakortis cfr. lita we obtained three highly degraded steroid derivatives, the known incisterol A2 (1) and the new incisterols A5 (2) and A6 (3). The new compounds were characterized basing on NMR and MS evidences along with comparison with model compounds. Incisterol A5 proved to bear a 17S-ethyl-15E,18-diene (incisterol numbering system) side chain, found for the first time in a marine organism. The new incisterols A5 and A6 proved to be potent inducers of transactivation of the pregnane X receptor (PXR) and they also stimulate the expression of CYP7A4 and MDR1 with a potency comparable to that of Rifaximin. These observations prompt to consider incisterols A5 and A6 as new potent agonists of PXR. On the other hand, the 17R-methyl analogue incisterol A2 shows only a poor PXR agonist activity. Molecular docking simulations elucidated the binding mechanism of the active incisterols in the ligand binding domain of PXR.

Bile acids exert genomic and nongenomic effects by interacting with membrane G-protein-coupled receptors, including the bile acid receptor GP-BAR1, and nuclear receptors, such as the farnesoid X receptor (FXR). These receptors regulate overlapping metabolic functions; thus, GP- BAR1/FXR dual agonists, by enhancing the biological response, represent an innovative strategy for the treatment of enter- oendocrine disorders. Here, we report the design, total synthesis, and in vitro/in vivo pharmacological evaluation of a new generation of dual bile acid receptor agonists, with the most potent compound, 19, showing promising pharmaco- logical profiles. We show that compound 19 activates GP- BAR1, FXR, and FXR regulated genes in the liver, increases the intracellular concentration of cAMP, and stimulates the release of the potent insulinotropic hormone GLP-1, resulting in a promising drug candidate for the treatment of metabolic disorders. We also elucidate the binding mode of the most potent dual agonists in the two receptors through a series of computations providing the molecular basis for dual GP-BAR1/FXR agonism.

Here, we report suvanine, a marine sponge sesterterpene, as an antagonist of the mammalian bile acid sensor farnesoid-X-receptor (FXR). Using suvanine as a template, we shed light on the molecular bases of FXR antagonism, identifying the essential conformational changes responsible for the transition from the agonist to the antagonist form. Molecular characterization of the nuclear corepressor NCoR and coactivator Src-1 revealed that receptor conformational changes are associated with a specific dynamic of recruitment of these cofactors to the promoter of OSTα, a FXR regulated gene. Using suvanine as a novel hit, a library of semisynthetic derivatives has been designed and prepared, leading to pharmacological profiles ranging from agonism to antagonism toward FXR. Deep pharmacological evaluation demonstrated that derivative 19 represents a new chemotype of FXR modulator, whereas alcohol 6, with a simplified molecular scaffold, exhibits excellent antagonistic activity.

Intervention in integrin-mediated cell adhesion and integrin signaling pathways is an ongoing area of research in medicinal chemistry and drug development. One key element in integrin-ligand interaction is the coordination of the bivalent cation at the metal ion-dependent adhesion site (MIDAS) by a carboxylic acid function, a consistent feature of all integrin ligands. With the exception of the recently discovered hydroxamic acids, all bioisosteric attempts to replace the carboxylic acid of integrin ligands failed. We report that phosphinates as well as monomethyl phosphonates represent excellent isosters, when introduced into integrin antagonists for the platelet integrin αIIbβ3. The novel inhibitors exhibit in vitro and ex vivo activities in the low nanomolar range. Steric and charge requirements of the MIDAS region were unraveled, thus paving the way for an in silico prediction of ligand activity and in turn the rational design of the next generation of integrin antagonists.

3-Substituted 6-phenyl-4H-imidazo[1,5-a][1,4]benzodiazepines and related compounds were synthesized as central benzodiazepine receptor (CBR) ligands. Most of the compounds showed high affinity for bovine and human CBR, their K(i) values spanning from the low nanomolar to the submicromolar range. In particular, imidazoester 5f was able to promote a massive flow of (36)Cl(-) in rat cerebrocortical synaptoneurosomes overlapping its efficacy profile with that of a typical full agonist. Compound 5f was then examined in mice for its pharmacological effects where it proved to be a safe anxiolytic agent devoid of the unpleasant myorelaxant and amnesic effects of the classical 1,4-benzodiazepines. Moreover, the selectivity of some selected compounds has been assessed in recombinant α(1)β(2)γ(2)L, α(2)β(1)γ(2)L, and α(5)β(2)γ(2)L human GABA(A) receptors. Finally, some compounds were submitted to molecular docking calculations along with molecular dynamics simulations in the Cromer's GABA(A) homology model.

Vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling in atherosclerosis and hypertension. Calcium-dependent signaling through calcium/calmodulin-dependent kinase II (CaMKII) and ERK1/2 activation plays an important role in the regulation of VSMC proliferation by agents such as α-adrenergic receptor agonists. Nevertheless, how the CaMKII and ERK pathways interact in VSMCs has yet to be characterized. The aim of the present study was to clarify this interaction in response to α1-adrenergic receptor-mediated VSMC proliferation. We discovered that phenylephrine stimulation resulted in complex formation between CaMKII and ERK in a manner that facilitated phosphorylation of both protein kinases. To assess the effects of CaMKII/ERK association on VSMC proliferation, we inhibited endogenous CaMKII either pharmacologically or by adenoviral-mediated gene transfer of a kinase-inactive CaMKII mutant. Inhibition of CaMKII activation but not CaMKII autonomous activity significantly decreased formation of the CaMKII/ERK complex. On the contrary, the expression of constitutively active CaMKII enhanced VSMC growth and CaMKII/ERK association. In addressing the mechanism of this effect, we found that CaMKII could not directly phosphorylate ERK but instead enhanced Raf1 activation. By contrast, ERK interaction with CaMKII facilitated CaMKII phosphorylation and promoted its nuclear localization. Our results reveal a critical role for CaMKII in VSMC proliferation and imply that CaMKII facilitates assembly of the Raf/MEK/ERK complex and that ERK enhances CaMKII activation and influences its subcellular localization.

Activated leukocyte cell adhesion molecule (ALCAM) plays a relevant role in tumor biology and progression. Our previous studies showed that ALCAM is expressed at the surface of epithelial ovarian cancer (EOC) cells and is released in a soluble form by ADAM-17-mediated shedding. This process is relevant to EOC cell motility and invasiveness, which is reduced by nonspecific inhibitors of ADAM-17. For this reason, ADAM-17 may represent a new useful target in anticancer therapy. Herein, we report the synthesis and biological evaluation of new ADAM-17 inhibitors containing an arylsulfonamidic scaffold. Among the new potential inhibitors, two very promising compounds 17 and 18 were discovered, with a nanomolar activity for ADAM-17 isolated enzyme. These compounds proved to be also the most potent in inhibiting soluble ALCAM release in cancer cells, showing a nanomolar activity on A2774 and SKOV3 cell lines.

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI-1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis-amidic moiety; 3) the introduction of a N-containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates. Molecular modeling studies for a deep binding-mode analysis of test compounds were also performed. The bis-carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI-1) and practically inactive against the lysine methyltransferase SET7/9.

Adenosine induces glioma cell proliferation by means of an antiapoptotic effect, which is blocked by cotreatment with selective A(3) AR antagonists. In this study, a novel series of N(2)-substituted pyrazolo[3,4-d]pyrimidines 2a-u was developed as highly potent and selective A(3) AR antagonists. The most performing compounds were derivatives 2a (R(1) = CH(3) and R(2) = COC(6)H(5); K(i) 334, 728, and 0.60 nM at the human A(1), A(2A), and A(3) ARs, respectively) and 2b (R(1) = CH(3) and R(2) = COC(6)H(4)-4-OCH(3); K(i) 1037, 3179, and 0.18 nM at the human A(1), A(2A), and A(3) ARs, respectively), which counteracted the effect of the A(3) AR agonists Cl-IB-MECA and IB-MECA on human glioma U87MG cell proliferation. This effect was concentration-dependent, with IC(50) values comparable to A(3) AR binding affinity values of 2a and 2b, thereby suggesting that their effects were receptor-mediated. Furthermore, the antiproliferative activity of the new compounds was demonstrated to be mediated by the block of A(3) AR agonist activation of intracellular kinases ERK 1/2.

We previously demonstrated that the gamma-glutamyl 16 amine derivative of vasoactive intestinal peptide (VIP) acts as structural VIP agonist with affinity and potency higher than VIP. Herein, we have evaluated the effects of VIP and gamma-Gln16-diaminopropane derivative of VIP (VIP-DAP3) on the proliferation and protection from oxidative stress induced by hydrogen peroxide (H2O2) on epidermoid carcinoma cell lines. We have found that 10(-11) M VIP-DAP3 completely antagonized the inhibition induced by H2O2 on both cell proliferation and S-phase distribution while these effects were only partially antagonized by equimolar concentrations of VIP. Moreover, both oxidative stress and intracellular lipid oxidation induced by H2O2 were reduced by VIP and completely antagonized by VIP-DAP3. Thereafter, we have found that H2O2 increased p38 kinase activity and both HSP70 and HSP27 expression. VIP and VIP-DAP3 again antagonized these effects partially or totally, respectively. H2O2 reduced the activity of extracellular signal-regulated kinases Erk-1/2 and Akt, signalling proteins involved in proliferation/survival pathways. Again VIP restored the activity of both kinases while VIP-DAP3 caused indeed an increase of their activity as compared to untreated cells. These data suggest that VIP-DAP3 has a stronger anti-oxidative activity as compared to VIP likely based on its super-agonistic binding on the putative receptor.

Ethyl 8-fluoro-6-(4-nitrophenyl)- and ethyl 8-fluoro-6-(3-nitrophenyl)-4 H-imidazo[1,5-a][1,4]benzodiazepine 3-carboxylate 6 and 7 were synthesized as central benzodiazepine receptor (CBR) ligands and tested for their ability to displace [(3)H]flumazenil from bovine and human cortical brain membranes. Both compounds showed high affinity for bovine and human CBR. In particular, compound 7 emerged as the most interesting compound, having a partial agonist profile in vitro while possessing useful activity in various animal models of anxiety. In accordance with its partial agonist profile, compound 7 was devoid of typical benzodiazepine side effects. The homology model of the GABA A receptor developed by Cromer et al. was used to assess the binding modes of ligands 6 and 7. From our docking results, the partial agonist activity elicited by compound 7 is likely to be due to the 3'-nitro substituent, which is in the appropriate position to interact with Thr193 of the gamma 2-subunit by means of a hydrogen bond.

The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signalling processes and cell proliferation. It has been reported that an improper amplification or activation of these enzymes is a distinctive feature of a number of human cancers, including breast cancers. Thus, the inhibition of Cdc25 phosphatases might provide a novel approach for the discovery of new and selective antitumor agents. By using the crystal structure of the catalytic domain of Cdc25B, structural models for the interaction of various Cdc25B inhibitors (1-13) with the enzyme were generated by computational docking. The parallel use of two efficient and predictive docking programs, AutoDock and GOLD, allowed mutual validation of the predicted binding poses. To evaluate their quality, the models were validated with known structure-activity relationships and site-directed mutagenesis data. The results provide an improved basis for structure-based ligand design and suggest a possible explanation for the inhibition mechanism of the examined Cdc25B ligands. We suggest that the recurring motif of a tight interaction between the inhibitor and the two arginine residues, 482 and 544, is of prime importance for reversible enzyme inhibition. In contrast, the irreversible inhibition mechanism of 1-4 seems to be associated with the close vicinity of the quinone ring and the Cys473 catalytic thiolate. We believe that this extensive study might provide useful hints to guide the development of new potent Cdc25B inhibitors as novel anticancer drugs. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

The angiotensin-converting enzyme II (ACE2) is a key molecular player in the regulation of vessel contraction, inflammation, and reduction of oxidative stress. In addition, ACE2 has assumed a prominent role in the fight against the COVID-19 pandemic-causing virus SARS-CoV-2, as it is the very first receptor in the host of the viral spike protein. The binding of the spike protein to ACE2 triggers a cascade of events that eventually leads the virus to enter the host cell and initiate its life cycle. At the same time, SARS-CoV-2 infection downregulates ACE2 expression especially in the lung, altering the biochemical signals regulated by the enzyme and contributing to the poor clinical prognosis characterizing the late stage of the COVID-19 disease. Despite its important biological role, a very limited number of ACE2 activators are known. Here, using a combined in silico and experimental approach, we show that ursodeoxycholic acid (UDCA) derivatives work as ACE2 activators. In detail, we have identified two potent ACE2 ligands, BAR107 and BAR708, through a docking virtual screening campaign and elucidated their mechanism of action from essential dynamics of the enzyme observed during microsecond molecular dynamics calculations. The in silico results were confirmed by in vitro pharmacological assays with the newly identified compounds showing ACE2 activity comparable to that of DIZE, the most potent ACE2 activator known so far. Our work provides structural insight into ACE2/ligand-binding interaction useful for the design of compounds with therapeutic potential against SARS-CoV-2 infection, inflammation, and other ACE2-related diseases.