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An agonist−antagonist switching strategy was performed to discover novel PPARα antagonists. Phenyldiazenyl derivatives of fibrates were developed, bearing sulfonimide or amide functional groups. A second series of compounds was synthesized, replacing the phenyldiazenyl moiety with amide or urea portions. Final compounds were screened by transactivation assay, showing good PPARα antagonism and selectivity at submicromolar concentrations. When tested in cancer cell models expressing PPARα, selected derivatives induced marked effects on cell viability. Notably, 3c, 3d, and 10e displayed remarkable antiproliferative effects in two paraganglioma cell lines, with CC50 lower than commercial PPARα antagonist GW6471 and a negligible toxicity on normal fibroblast cells. Docking studies were also performed to elucidate the binding mode of these compounds and to help interpretation of SAR data.

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases known to exert multiple regulatory roles in tumor progression. A variety of chemical classes have been explored for targeting individual MMP isoforms. In the present study, we further developed our isatin based scaffold BB0223107 capable of binding to and inactivating MMP-2 in a zinc-independent manner (Agamennone et al., 2016). Forty four new compounds were synthesized based on the modified BB0223107. All compounds were tested in enzyme inhibition assays against MMP-2, −8 and −13. SAR studies demonstrated that 5-het(aryl)-3-aminoindolin-2-ones (37–39) were active toward MMP-2 and MMP-13. The most potent compounds 33 and 37 displayed an IC50 of 3 µM against MMP-13 and showed a negligible activity toward MMP-8; almost all new compounds were inactive toward MMP-8. Replacement of the isatin ring with a biaryl system (compound 33) did not decrease the potency against MMP-13 but reduced the selectivity. Structure-based computational studies were carried out to rationalize the inhibitory activity data. The analysis of binding geometries confirmed that all fragments occupied the S1′ site in the three enzymes while no ligand was able to bind the catalytic zinc ion. To the best of our knowledge, this is the first example of 3-aminoindolin-2-one-based MMP inhibitors that, based on the computer modeling study, do not coordinate the zinc ion. Thus, the het(aryl)-3-aminoindolin-2-one derivatives emerge as a drug-like and promising chemotype that, along with the hetaryl variations, represents an alternative and thrifty tool for chemical space exploration aimed at MMP inhibitor design.

The reduced activation of PPARs has a positive impact on cancer cell growth and viability in multiple preclinical tumor models, suggesting a new therapeutic potential for PPAR antagonists. In the present study, the benzothiazole amides 2a-g were synthesized and their activities on PPARs were investigated. Transactivation assay showed a moderate activity of the novel compounds as PPARα antagonists. Notably, in cellular assays they exhibited cytotoxicity in pancreatic, colorectal and paraganglioma cancer cells overexpressing PPARα. In particular, compound 2b showed the most remarkable inhibition of viability (greater than 90%) in two paraganglioma cell lines, with IC50 values in the low micromolar range. In addition, 2b markedly impaired colony formation capacity in the same cells. Taken together, these results show a relevant anti-proliferative potential of compound 2b, which appears particularly effective in paraganglioma, a rare tumor poorly responsive to chemotherapy.

[Image: see text] The development of PPARα/γ dual or PPARα/γ/δ pan-agonists could represent an efficacious approach for a simultaneous pharmacological intervention on carbohydrate and lipid metabolism. Two series of new phenyldiazenyl fibrate derivatives of GL479, a previously reported PPARα/γ dual agonist, were synthesized and tested. Compound 12a was identified as a PPAR pan-agonist with moderate and balanced activity on the three PPAR isoforms (α, γ, δ). Moreover, docking experiments showed that 12a adopts a different binding mode in PPARγ compared to PPARα or PPARδ, providing a structural basis for further structure-guided design of PPAR pan-agonists. The beneficial effects of 12a were evaluated both in vitro, on the expression of PPAR target key metabolic genes, and ex vivo in two rat tissue inflammatory models. The obtained results allow considering this compound as an interesting lead for the development of a new class of PPAR pan-agonists endowed with an activation profile exploitable for therapy of metabolic syndrome.

Compounds containg catechol or bisphosphonate were tested as inhibitors of the zinc metalloproteases, thermolysin (TLN), pseudolysin (PLN) and aureolysin (ALN) which are bacterial virulence factors, and the human matrix metalloproteases MMP-9 and −14. Inhibition of virulence is a putative strategy in the development of antibacterial drugs, but the inhibitors should not interfere with human enzymes. Docking indicated that the inhibitors bound MMP-9 and MMP-14 with the phenyl, biphenyl, chlorophenyl, nitrophenyl or methoxyphenyl ringsystem in the S1′-subpocket, while these ringsystems entered the S2′- or S1 -subpockets or a region involving amino acids in the S1′- and S2′-subpockets of the bacterial enzymes. An arginine conserved among the bacterial enzymes seemed to hinder entrance deeply into the S1′-subpocket. Only the bisphosphonate containing compound RC2 bound stronger to PLN and TLN than to MMP-9 and MMP-14. Docking indicated that the reason was that the conserved arginine (R203 in TLN and R198 in PLN) interacts with phosphate groups of RC2., Graphical Abstract

Alzheimer&rsquo, s disease (AD) is generally recognized as a multifactorial neurodegenerative pathology with an increasing impact on society. Tenuazonic acid (TA) is a natural compound that was recently identified as a potential multitarget ligand with anti-cholinesterase, anti-amyloidogenic and antioxidant activities. Using its structure as a chemical scaffold, we synthesized and evaluated new derivatives (1&ndash, 5), including tenuazonic-donepezil (TA-DNP) hybrids (4 and 5) due to the clinical importance of the anti-AD drug donepezil. These novel compounds all achieved activity in the micromolar range towards all selected targets and demonstrated to be potentially orally absorbed. Moreover, a selected compound (1) was further investigated as a chelating agent towards copper (II), zinc (II) and iron (III) and showed good chelating ability (pFe = 16.6, pCu = 11.6, pZn = 6.0 at pH 7.4). Therefore, the TA motif can be considered an interesting building block in the search for innovative multi-functional anti-neurodegenerative drugs, as exemplified by hybrid 5, a promising non-cytotoxic lead compound adequate for the early stages of AD, and capable of ameliorating the oxidative status of SH-SY5Y human neuroblastoma cells.

Matrix Metalloproteinases (MMPs) are a family of secreted and membrane-bound enzymes, of which 24 isoforms are known in humans. These enzymes degrade the proteins of the extracellular matrix and play a role of utmost importance in the physiological remodeling of all tissues. However, certain MMPs, such as MMP-2, -9, and -13, can be overexpressed in pathological states, including cancer and metastasis. Consequently, the development of MMP inhibitors (MMPIs) has been explored for a long time as a strategy to prevent and hinder metastatic growth, but the important side effects linked to promiscuous inhibition of MMPs prevented the clinical use of MMPIs. Therefore, several strategies were proposed to improve the therapeutic profile of this pharmaceutical class, including improved selectivity toward specific MMP isoforms and targeting of specific organs and tissues. Combining both approaches, we conducted the synthesis and preliminary biological evaluation of a series of (2-aminobenzothiazole)-methyl-1,1-bisphosphonic acids active as selective inhibitors of MMP-13 via in vitro and in silico studies, which could prove useful for the treatment of bone metastases thanks to the bone-targeting capabilities granted by the bisphosphonic acid group.

Matrix metalloproteinases (MMPs) are zinc-dependent hydrolytic enzymes of great biological relevance, and some of them are key to the neuroinflammatory events and the brain damage associated to stroke. Non-zinc binding ligands are an emerging trend in drug discovery programs in this area due to their lower tendency to show off-target effects. 7-Amino-phenanthridin-6-one is disclosed as a new framework able to inhibit matrix metalloproteinases by binding to the distal part of the enzyme S1' site, as shown by computational studies. A kinetic study revealed inhibition to be noncompetitive. Some of the compounds showed some degree of selectivity for the MMP-2 and MMP-9 enzymes, which are crucial for brain damage associated to ischemic stroke. Furthermore, some compounds also had a high neuroprotective activity against oxidative stress, which is also very relevant aspect of ischaemic stroke pathogenesis, both decreasing lipid peroxidation and protecting against the oxidative stress-induced reduction in cell viability. One of the compounds, bearing a 2-thienyl substituent at C-9 and a 4-methoxyphenylamino at C-7, had the best-balanced multitarget profile and was selected as a lead on which to base future structural manipulation.

Bisphosphonates such as zoledronic, alendronic and risedronic acids are a class of drugs clinically used to prevent bone density loss and osteoporosis. Novel P-C-P bisphosphonates were synthesized for targeting human farnesyl pyrophosphate synthase (hFPPS) and human geranylgeranyl pyrophosphate synthase (hGGPPS), key enzymes of the mevalonate pathway, and capable of anti-proliferative action on a number of cell lines (PC3, MG63, MC3T3, RAW 264.7, J774A.1, bone marrow cells and their co-colture with PC3) involved in bone homeostasis, bone formation and death. Among sixteen compounds, [1-hydroxy-2-(pyrimidin-2-ylamino)ethane-1,1-diyl]bis(phosphonic acid) (10) was effective in reducing PC3 and RAW 264.7 cell number in crystal-violet and cell-dehydrogenase activity assays at 100 μM concentration. 10 reduced differentiated osteoclasts number similarly with zoledronic acid in osteoclastogenesis assay. At nanomolar concentrations, 10 was more effective than zoledronic acid in inducing mineralization in MC3T3 and murine bone marrow cells. Further, 10 significantly inhibited the activity of hFPPS showing an IC50 of 0.31 μM and a remarkable hydroxyapatite binding of 90%. Docking calculations were performed identifying putative interactions between some representative novel bisphosphonates and both hFPPS and hGGPPS. Then, 10 was found to behave similarly or even better than zoledronic acid as a anti-resorptive agent.

Matrix metalloproteinase 2 (MMP-2) is a well-known anticancer target belonging to the MMP family. Because of the bilateral role of MMPs in cancer, developing highly selective MMP-2 inhibitors is a current challenge. In this paper, we investigated the binding modes of green tea polyphenols epigallocatechin gallate and epicatechin into the active site of the MMP-2 enzyme. The structure-based analysis allowed the optimization of these hits and hence led to a better lead candidate. Moreover, using a pharmacophore model, we screened FooDB compounds and selected food components as potential MMP-2 inhibitors. The search for food-derived compounds that target this enzyme may represent a strategy to identify new lead molecules with improved safety profiles and provide indications about possible functional foods.

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases known to exert multiple regulatory roles in tumor progression and invasiveness. This encouraged over the years the approach of MMP, and particularly MMP-2, targeting for anticancer treatment. Early generations of MMP inhibitors, based on aspecific zinc binding groups (ZBGs) assembled on (pseudo)peptide scaffolds, have been discontinued due to the clinical emergence of toxicity and further drawbacks, giving the way to inhibitors with alternative zinc-chelator moieties or not binding the catalytic zinc ion. In the present paper, we continue the search for new non-zinc binding MMP-2 inhibitors: exploiting previously identified compounds, a virtual screening (VS) campaign was carried out and led to the identification of a new class of ligands. The structure-activity relationship (SAR) of the benzimidazole scaffold was explored by synthesis of several analogues whose inhibition activity was tested with enzyme inhibition assays. By performing the molecular simplification approach, we disclosed different sets of single-digit micromolar inhibitors of MMP-2, with up to a ten-fold increase in inhibitory activity and ameliorated selectivity towards off-target MMP-8, compared to selected lead compound. Molecular dynamics calculations conducted on complexes of MMP-2 with docked privileged structures confirmed that analyzed inhibitors avoid targeting the zinc ion and dip inside the S1′ pocket. Present results provide a further enrichment of our insights for the design of novel MMP-2 selective inhibitors.

Over the course of the past decade, peroxisome proliferator-activated receptors (PPARs) have been identified as part of the cannabinoid signaling system: both phytocannabinoids and endocannabinoids are capable of binding and activating these nuclear receptors. Fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and other N-acylethanolamines. These substances have been shown to have numerous anticancer effects, and indeed the inhibition of FAAH has multiple beneficial effects that are mediated by PPARα subtype and by PPARγ subtype, especially antiproliferation and activation of apoptosis. The substrates of FAAH are also PPAR agonists, which explains the PPAR-mediated effects of FAAH inhibitors. Much like cannabinoid ligands and FAAH inhibitors, PPARγ agonists show antiproliferative effects on cancer cells, suggesting that additive or synergistic effects may be achieved through the positive modulation of both signaling systems. In this Miniperspective, we discuss the development of novel FAAH inhibitors able to directly act as PPAR agonists and their promising utilization as leads for the discovery of highly effective anticancer compounds.

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis playing a central role in cardiovascular disease, obesity, and diabetes. These receptors show a high degree of stereoselectivity towards several classes of drugs. This review covers the most relevant findings that have been made in the last decade and takes into consideration only those compounds in which stereochemistry led to unexpected results or peculiar interactions with the receptors. These cases are reviewed and discussed with the aim to show how enantiomeric recognition originates at the molecular level. The structural characterization by crystallographic methods and docking experiments of complexes formed by PPARs with their ligands turns out to be an essential tool to explain receptor stereoselectivity.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases, capable to degrade the extracellular matrix (ECM) in physiologic conditions. Because of their overexpression and pivotal role in many pathological events, they have been proposed as a therapeutic and prognostic target for a number of diseases. Selectivity among MMPs is essential for realizing the clinical potential of inhibitors. The design of MMP inhibitors (MMPIs) has largely focused on development of various compounds containing a zinc binding group (ZBG) in their structure, with hydroxamate being the most potent one. Due to the high degree of homology in the catalytic domain of all the MMPs, the specificity and selectivity of first generation hydroxamate MMPIs were minimal, with several off-target effects and binding to other metzincins. This review highlights the role of phosphonate as ZBG in the design and development of new MMPIs.

Zinc (II) is an important biometal in human physiology. Moreover, in the last two decades, it was deeply studied for its involvement in several pathological states. In particular, the regulation of its concentration in synaptic clefts can be fundamental for the treatment of neurodegenerative diseases, such as Alzheimer’s disease (AD). Zinc (II) is also a constituent of metalloenzymes (i.e., matrix metalloproteinases, MMPs, and carbonic anhydrases, CAs) with catalytic function; therefore, it can be an important target for the inhibition of these proteins, frequently involved in cancer onset. This review is focused on the significance of zinc (II) chelating agents in past and future medicinal chemistry research, and on the importance of selectivity in order to revamp the possibility of their use in therapy, often hindered by possible side effects.

Periodontitis is an inflammatory disease caused by anaerobic bacteria, including Porphyromonas gingivalis. Lipopolysaccharide (LPS)‑stimulated persistent inflammation is responsible for an increase in matrix metalloproteinase (MMP) expression, resulting in periodontal tissue destruction. The aim of the present study was to investigate synthesized bisphosphonic MMP inhibitors, in an in vitro model consisting of human gingival fibroblasts exposed to LPS, and to compare the biological responses to those induced by zoledronate (ZA), a commercial bisphosphonate. MTT and lactate dehydrogenase (LDH) assays were used to measure cell viability and cytotoxicity, respectively. ELISA was performed to evaluate prostaglandin E2 (PGE2), interleukin (IL)6 and collagen secretion, while western blotting was used to analyze MMP expression. No effect on viability and low cytotoxicity were observed following treatment with bisphosphonate compounds. In the present study, treatment with compound 1 did not increase the release of PGE2 and IL6. Increased levels of collagen I secretion were reported when compound 3 and ZA were administered. An increase of MMP8 was observed following ZA treatment, while a decrease of MMP9 and MMP14 following treatment with compounds 1, 2 and ZA were reported. The performance of compound 1 was optimal in terms of cell viability. Compound 1 also did not induce inflammation, and had the ability to counteract LPS‑induced increases in MMP expression. These data suggested that compound 1 was the most suitable treatment to progress to an in vivo animal study, with the aim to confirm its use for the treatment of periodontitis.

A new series of derivatives of the PPAR alpha/gamma dual agonist 1 allowed us to identify the ligand (S)-6 as a potent partial agonist of both PPAR alpha and gamma subtypes. X-ray studies in PPAR gamma revealed two different binding modes of (S)-6 to the canonical site. However, (S)-6 was also able to bind an alternative site as demonstrated by transactivation assay in the presence of a canonical PPAR gamma antagonist and supported from docking experiments. This compound did not activate the PPAR gamma-dependent program of adipocyte differentiation inducing a very less severe lipid accumulation compared to rosiglitazone but increased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Finally, (S)-6 inhibited the Cdk5-mediated phosphorylation of PPAR gamma at serine 273 that is currently considered the mechanism by which some PPAR gamma partial agonists exert antidiabetic effects similar to thiazolidinediones, without showing their typical side effects. This is the first PPAR alpha/gamma dual agonist reported to show this inhibitory effect representing the potential lead of a new class of drugs for treatment of dyslipidemic type 2 diabetes.

Metaglidasen is a fibrate-like drug reported as a selective modulator of peroxisome proliferator-activated receptor γ (PPARγ), able to lower plasma glucose levels in the absence of the side effects typically observed with thiazolidinedione antidiabetic agents in current use. Herein we report an improved synthesis of metaglidasen's metabolically active form halofenic acid (R)-2 and that of its enantiomer (S)-2. The activity of the two stereoisomers was carefully examined on PPARα and PPARγ subtypes. As expected, both showed partial agonist activity toward PPARγ; the investigation of PPARα activity, however, led to unexpected results. In particular, (S)-2 was found to act as a partial agonist, whereas (R)-2 behaved as an antagonist. X-ray crystallographic studies with PPARγ were carried out to gain more insight on the molecular-level interactions and to propose a binding mode. Given the adverse effects provoked by fibrate drugs on skeletal muscle function, we also investigated the capacity of (R)-2 and (S)-2 to block conductance of the skeletal muscle membrane chloride channel. The results showed a more beneficial profile for (R)-2, the activity of which on skeletal muscle function, however, should not be overlooked in the ongoing clinical trials studying its long-term effects.

Purpose. To investigate the correlation between best-corrected visual acuity (BCVA), the foveal inner segment/outer segment (IS/OS) junction or ellipsoid portion of inner segment (EPIS/ellipsoid zone), and the cone outer segment tips (COST) line or interdigitation zone integrity in eyes with uveitic macular edema (ME).Method. A retrospective observational study involving all patients from January 2012 to December 2013 with uveitic ME was performed. All patients underwent BCVA using Snellen charts spectral-domain optical coherence tomography (SD-OCT) examination using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany).Results. Fifty-two eyes from 45 patients were included in this study. Multivariate analysis showed a negative correlation between BCVA and the central retinal subfield thickness (CST), the cystoid pattern of edema, and the interdigitation zone interruption. Univariate logistic analysis showed a strong correlation between the ellipsoid zone and the interdigitation zone integrity.Conclusions. The ellipsoid zone defect, the interdigitation zone interruption, and the CST are correlated with poor vision. Visual acuity is also strongly affected by the cystoid pattern. The interdigitation zone integrity appears to be the most important factor in the visual prognosis of uveitic ME.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARalpha/gamma dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARalpha and fine-tuned moderate activity on PPARgamma. For the most interesting compound (S)-3, docking studies in PPARalpha and PPARalpha provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARalpha/gamma activity which probably involves a synergistic effect on both receptor subtypes.

A series of nanomolar phosphonate matrix metalloproteinase (MPP) inhibitors was tested for inhibitory activity against a panel of selected human carbonic anhydrase (CA, EC 4.2.1.1) isozymes, covering the cancer-associated CA IX and XII. None of the reported sulfonyl and sulfonylamino-derivatives sensitively affected the catalytic activity of the cytosolic isoforms CA I and II, which are considered off-target isoforms in view of their physiological role. The most active inhibitors were in the series of chiral N-(sulfonyl)phosphovaline derivatives, which showed good to excellent inhibitory activity over target CAs, with compound 15 presenting the best isoform-selectivity toward CA IX. We suggest here that the phosphonates have the potential as dual inhibitors of MMPs and CAs, both involved in tumor formation, invasion and metastasis.

PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.

We screened a short series of new chiral diphenylmethane derivatives and identified potent dual PPARα/γ partial agonists. As both enantiomers of the most active compound 1 displayed an unexpected similar transactivation activity, we performed docking experiments to provide a molecular understanding of their similar partial agonism. We also evaluated the ability of both enantiomers of 1 and racemic 2 to inhibit colorectal cancer cells proliferation: (S)-1 displayed a more robust activity due, at least in part, to a partial inhibition of the Wnt/β-catenin signalling pathway that is upregulated in the majority of colorectal cancers. Finally, we investigated the effects of (R)-1, (S)-1 and (R,S)-2 on mitochondrial function and demonstrated that they activate the carnitine shuttle system through upregulation of carnitine/acylcarnitine carrier (CAC) and carnitine-palmitoyl-transferase 1 (CPT1) genes. Consistent with the notion that these are PPARα target genes, we tested and found that PPARα itself is regulated by a positive loop. Moreover, these compounds induced a significant mitochondrial biogenesis. In conclusion, we identified a new series of dual PPARα/γ agonists endowed with novel anti-proliferative properties associated with a strong activation of mitochondrial functions and biogenesis, a potential therapeutic target of the treatment of insulin resistance.

Inflammatory choroidal neovascularization is a severe but uncommon complication of uveitis, more frequent in posterior uveitis such as punctate inner choroidopathy, multifocal choroiditis, serpiginous choroiditis, and Vogt-Koyanagi-Harada syndrome. Its pathogenesis is supposed to be similar to the wet age related macular degeneration: hypoxia, release of vascular endothelial growth factor, stromal cell derived factor 1-alpha, and other mediators seem to be involved in the uveitis-related choroidal neovascularization. A review on the factors implicated so far in the pathogenesis of inflammatory choroidal neovascularization was performed. Also we reported the success rate of single studies concerning the therapies of choroidal neovascularization secondary to uveitis during the last decade: photodynamic therapy, intravitreal bevacizumab, and intravitreal ranibizumab, besides steroidal and immunosuppressive therapy. Hereby a standardization of the therapeutic approach is proposed.

The complexity of matrix metalloproteinase inhibitors (MMPIs) design derives from the difficulty in carefully addressing their inhibitory activity towards the MMP isoforms involved in many pathological conditions. In particular, specific metalloproteinases, such as MMP-2 and MMP-9, are key regulators of the ‘vicious cycle’ occurring between tumor metastases growth and bone remodeling. In an attempt to devise new approaches to selective inhibitor derivatives, we describe novel bisphosphonate bone seeking MMP inhibitors (BP-MMPIs), capable to be selectively targeted and to overcome undesired side effects of broad spectrum MMPIs. In vitro activity (IC50 values) for each inhibitor was determined against MMP-2, -8, -9 and -14, because of their relevant role in skeletal development and renewal. The results show that BP-MMPIs reached IC50 values of enzymatic inhibition in the low micromolar range. Computational studies, used to rationalize some trends in the observed inhibitory profiles, suggest a possible differential binding mode in MMP-2 that explains the selective inhibition of this isoform. In addition, survival assay was conducted on J774 cell line, a well known model system used to evaluate the structure–activity relationship of BPs for inhibiting bone resorption. The resulting data, confirming the specific activity of BP-MMPIs, and their additional proved propensity to bind hydroxyapatite powder in vitro, suggest a potential use of BP-MMPIs in skeletal malignancies.

PPARs are nuclear receptors with a critical physiological role in lipid and glucose metabolism. As part of our effort to develop new and selective PPAR agonists containing stilbene and its bioisoster phenyldiazene, novel analogs were synthesized starting from tyrosine and evaluated as PPAR agonists. We tested the effects of phenyloxazole replacement of GW409544, a well-known PPARalpha/gamma dual agonist, with stilbene or phenyldiazene moiety, spaced by an ether bridge to tyrosine portion. These structural modifications provided potent and selective PPARgamma agonists. Molecular docking studies performed on these new compounds complemented the experimental results and allowed to gain some insights into the nature of binding of the ligands.

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in several pathological states. Among them, MMP-2 is a relevant therapeutic target because of its role in cancer development and progression. Many MMP inhibitors (MMPIs) have been discovered over the last 30 years, and the majority of them contain a functional group that binds the zinc ion (zinc-binding group; ZBG). Unfortunately, no MMPIs have reached the market yet, owing to toxic effects due to unselective interactions of the ZBG. The new generation of MMPIs that do not bind the zinc ion could overcome problems of selectivity and toxicity, but have so far been developed only for MMP-8, -12, and -13. In this work, a virtual screening protocol was established by combining ligand- and structure-based methods to identify non-zinc-binding MMP-2 inhibitors using a new-generation MMP-8 inhibitor as a probe to find unexplored interactions in the MMP-2 S1' site. The screening allowed the identification of micromolar MMP-2 inhibitors that putatively avoid binding the zinc ion, as demonstrated by docking calculations. The LIA model, built to correlate predicted and experimental binding energies of the identified non-zinc-binding MMP-2 hits, underpins the reliability of the predicted docking poses.

2 H -1,4-Benzoxazine amidine derivatives are drugs acting as modulators of the skeletal muscle and pancreatic beta cell ATP-sensitive-K + (KATP) channels. With the aim of evaluating the influence of absolute configuration on the biological activity of these drugs, we herein report the optimization of a synthetic route to obtain both enantiomers of some of these compounds with improved chemical yield and high enantiomeric excess.

Matrix metalloproteinases (MMPs) are attractive biological targets that play a key role in many physiopathological processes such as degradation of extracellular matrix proteins, release and cleavage of cell-surface receptors, tumour progression, homeostatic regulation and innate immunity. A series of 5-hydroxy, 5-substituted pyrimidine-2,4,6-triones were rationally designed, prepared and tested as inhibitors of gelatinases MMP-2 and MMP-9 and collagenase MMP-8. On one side, the presence of the 5-hydroxyl group, that represents an typical feature of this class of compounds, ensured an attractive pharmacokinetic profile while on the other suitably substituted biaryl molecular fragments, attached to position 5 through a ketomethylene linker, guaranteed favourable interaction in the deep region of the S(1)' enzymatic subsite. This rational design led to the discovery of highly potent MMP inhibitors. In particular, biphenyl derivatives bearing at the para position COCH(3) and OCF(3) substituents permitted to inhibit gelatinases MMP-2 and MMP-9, with IC(50) values as low as 30 nM and 21 nM, respectively, whereas the introduction at the same position of the bulkier SO(2)CH(3) group afforded a potent collagenase MMP-8 inhibitor with an IC(50) value equal to 66 nM. Molecular docking simulations allowed us to elucidate key interactions driving the binding of the top active compounds towards their preferred MMP target.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.

Matrix metalloproteinases (MMPs) are well-established targets for several pathologies. In particular, MMP-2 and MMP-13 play a prominent role in cancer progression. In this study, a structure-based screening campaign was applied to prioritize metalloproteinase-oriented fragments. This computational model was applied to a representative fragment set from the publically available EDASA Scientific compound library. These fragments were prioritized, and the top-ranking hits were tested in a biological assay to validate the model. Two scaffolds showed consistent activity in the assay, and the isatin-based compounds were the most interesting. These latter fragments have significant potential as tools for the design and realization of novel MMP inhibitors. In addition to their micromolar activity, the chemical synthesis affords flexible and creative access to their analogues.

A small library of 2,3-dihydroxybenzamide- and N-(2,3-dihydroxyphenyl)-4-sulfonamide-based microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors was identified following a step-by-step optimization of small aromatic fragments selected to interact in focused regions in the active site of mPGES-1. During the virtual optimization process, the 2,3-dihydroxybenzamide moiety was first selected as a backbone of the proposed new chemical entities; the identified compounds were then synthesized and biologically evaluated, identifying derivatives with very promising inhibitory activities in the micromolar range. Subsequent structure-guided replacement of the 2,3-dihydroxybenzamide by the N-(2,3-dihydroxyphenyl)sulfonamide moiety led to the identification of N-(2,3-dihydroxyphenyl)-4-biphenylsulfonamide (6), the most potent small molecule of the series (IC50 =0.53 ± 0.04 μM). The simple synthetic procedure and the possibility of enhancing the potency of this class of inhibitors through additional structural modifications pave the way for further development of new molecules with mPGES-1-inhibitory activity, with potential application as anti-inflammatory and anticancer agents.

A series of saponins and sapogenins from Medicago species were tested for their ability to bind and activate the nuclear receptor PPARγ by SPR experiments and transactivation assay, respectively. The SPR analysis proved to be a very powerful and fast technique for screening a large number of compounds for their affinity to PPARγ and selecting the better candidates for further studies. Based on the obtained results, the sapogenin caulophyllogenin was proved to be a partial agonist towards PPARγ and the X-ray structure of its complex with PPARγ was also solved, in order to investigate the binding mode in the ligand binding domain of the nuclear receptor. This is the first known crystal structure of a sapogenin directly interacting with PPARγ. Another compound of the series, the echinocistic acid, showed antagonist activity towards PPARγ, a property that could be useful to inhibit the adipocyte differentiation which is a typical adverse effect of PPARγ agonists. This study confirms the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating the metabolic syndrome.

A number of matrix metalloproteinases (MMPs), proteins important in the balance of bone remodeling, play a critical role both in cancer metastasis and in bone matrix turnover associated with the presence of cancer cells in bone. Here, we report the synthesis and biological evaluation of a new class of MMP inhibitors characterized by a bisphosphonate function as the zinc binding group. Since the bisphosphonate group is also implicated in osteoclast inhibition and provides a preferential affinity to biological apatite, the new molecules can be regarded as bone-seeking medicinal agents. Docking experiments were performed to clarify the mode of binding of bisphosphonate inhibitors in the active site of MMP-2. The most promising of the studied bisphosphonates showed nanomolar inhibition against MMP-2 and resulted in potent inhibition of osteoclastic bone resorption in vitro.

Convenient synthetic routes to KN-93, N-(2-{[[(2 E)-3-(4-chlorophenyl)prop-2-enyl](methyl)amino]methyl}phenyl)- N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide,a well-known Ca 2 +/calmoduline-dependentprotein kinase II (CaMKII) inhibitor, are described. The methodsproposed start from easily available reagents- and allowready preparation of the final compound in moderate- overallyields. Most of the synthetic steps proposed were microwave assisted.

Chiral chromatographic resolution of a series of dual PPARα/γ agonists was performed on the commercially available Chiralcel OD and Chiralpak AD columns in normal phase mode using a mobile phase consisting of a mixture of n-hexane, 2-propanol and trifluoroacetic acid at constant volume ratio. The selected CSPs resolved most compounds as underivatized acids without requiring time consuming analysis. Both electronic features and steric hindrance affected chiral interactions between the analytes and the CSPs. Additional information on the chiral recognition mechanism was deduced from the chromatographic behaviour of some selected methyl esters.

The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPAR alpha/gamma dual agonist 1, allowed the identification of new ligands with improved potency on PPAR alpha and unchanged activity on PPAR gamma. For the most interesting stereoisomers S-2 and S-4, X-ray studies in PPAR gamma and docking experiments in PPAR alpha provided a molecular explanation for their different behavior as full and partial agonists of PPAR alpha and PPAR gamma, respectively. Due to the adverse effects provoked by hypolipidemic drugs on skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle membrane chloride channel conductance and found that these ligands have a pharmacological profile more beneficial compared to fibrates currently used in therapy.

The LC enantiomeric separation of several dual PPARα/γ agonists on the commercially available Chiralcel OD and Chiralpak AD columns has been evaluated in normal phase mode using a mobile phase consisting in a mixture of n-hexane, 2-propanol and trifluoroacetic acid at constant volume ratio. Most compounds were separated as underivatized acids without requiring time consuming analysis. Some complementary selectivity was evidenced on the two investigated chiral stationary phases related to the different accessibility of the active sites of the helical cavities. Additional information on the chiral recognition mechanism were deduced from the chromatographic behaviour of some selected methyl esters.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.

To evaluate N -hydroxyurea as zinc binding group in the design of MMP inhibitors, two peptidyl 1-hydroxyureas were prepared by N -hydroxycarbamoylation of the diastereomeric dipeptides H-Leu-Phe-NHMe and H- d -Leu-Phe-NHMe. Peptidyl 1-hydroxyureas were more potent than the parent peptides, but dramatically weaker (4–5 orders of magnitude) than the isosteric ( R )-succinylhydroxamate analogue, which displays IC 50 in the range of nM vs MMP-1, -3, -7 and sub-nM vs MMP-2, -8, and -9. The peptidyl 1-hydroxyurea 1a attained an IC 50 of 20 μM vs MMP-9, and substantially approaches inhibition of known N -hydroxyureas based on aminoacids or peptides against other zinc metalloenzymes and non-peptidic N -hydroxyureas against MMPs. Strong preference of the O–N1–C O unit for the antiperiplanar amide bond conformation seems to be the major limit for more effective zinc chelation. Methylation of a peptidyl 1-hydroxyurea at N3, to promote the synperiplanar O–N1–C O conformation required for zinc chelation and improve affinity, resulted in release of a methylimidazolidine-2,4-dione through an undesired intramolecular reaction reminiscent of the Edman peptide degradation.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose and lipid metabolism. They are activated by natural ligands, such as fatty acids, and are also targets of synthetic antidiabetic and hypolipidemic drugs. By using cell-based reporter assays, we studied the transactivation activity of two enantiomeric ureidofibrate-like derivatives. In particular, we show that the R-enantiomer, (R)-1, is a full agonist of PPARgamma, whereas the S-enantiomer, (S)-1, is a less potent partial agonist. Most importantly, we report the x-ray crystal structures of the PPARgamma ligand binding domain complexed with the R- and the S-enantiomer, respectively. The analysis of the two crystal structures shows that the different degree of stabilization of the helix 12 induced by the ligand determines its behavior as full or partial agonist. Another crystal structure of the PPARgamma.(S)-1 complex, only differing in the soaking time of the ligand, is also presented. The comparison of the two structures of the complexes with the partial agonist reveals significant differences and is suggestive of the possible coexistence in solution of transcriptionally active and inactive forms of helix 12 in the presence of a partial agonist. Mutation analysis confirms the importance of Leu(465), Leu(469), and Ile(472) in the activation by (R)-1 and underscores the key role of Gln(286) in the PPARgamma activity.

Bisphosphonates (BPs) are drugs clinically used in resorptive diseases. It was already proved that some clinically relevant BPs can inhibit a class of enzymes called matrix metalloproteinases (MMPs), required during tissue remodelling. Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present study was to compare the effect of zoledronic acid (ZA), the most potent bisphosphonate available as therapy, with new sulfonamide containing BPs in an in vitro model of human gingival fibroblasts (HGFs). Western blot was used to measure procollagen I, β1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability; L-lactate-dehydrogenase (LDH) measurement was performed for toxicity evaluation and ELISA for prostaglandin E2 (PGE2) secretion assessment. When compared with ZA, the treatment with the newly synthesized compounds shows increasing viability, procollagen I expression and decreased expression of β1 integrin in HGFs. Higher levels of released LDH, PGE2 and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthesized compounds-treated samples. These findings allowed to conclude that new tested BPs did not affect HGFs viability and adhesion, did not induce cellular toxicity, were not responsible for inflammatory event induction and could preserve the physiological matrix turnover. It could be hypothesized that the new molecules were better tolerated by soft tissues, resulting in lesser side effects.

Matrix metalloproteinases (MMPs) are zinc enzymes responsible for the degradation of the extracellular matrix. With this function, MMPs are involved in many physiological processes, but also in many pathological states. MMP-13 is implicated in the degradation of type II collagen, the main structural protein of articular cartilage, contributing to the development of osteoarthritis and inflammatory diseases. In the last years, a new generation of potent and selective MMP inhibitors (MMPIs) has been identified and classified as non-zinc-binding inhibitors (NZBIs). Several MMP-13 NZBIs have been developed and crystallographically determined in complex with the enzyme. Here, we provide a detailed review of the current knowledge about this class of MMP-13 inhibitors and, by using computational procedures, we highlight the molecular determinants that are needed for the binding process. In particular, FLAP, a program based on GRID molecular interaction fields, was used to analyze the ligand-protein interactions: molecular shape and hydrogen bond acceptor groups strongly influence the binding according to the ligand-based modeling, while the aromatic interactions are better identified by the structure-based study. The complementary results can be combined in a high performance model, showing the effectiveness of molecular interaction field based approaches to search for novel MMP-13 NZBIs.

Enantiomerically pure (R)-2-chloro-1-(pyridin-3-yl)ethanol has been prepared by kinetic resolution performed in the presence of Candida antarctica SP435-L lipase immobilized on a macroporous polyacrylate resin (Novozym 435®). It was converted into (R)-1-(pyridin-3-yl)-2-aminoethanol, left-hand side of β3-adrenergic receptor ligands.

The phosphotryptophan derivative l-Pro-l-Leu-l-(P)Trp(OH)(2) (2b) was reported as the first example of left-hand-sideLeft-hand-side inhibitors: inhibitors that bind in the unprime region of the enzyme active site, in reference to the convention of drawing the unprimed residues of a peptide substrate on the left side. [R.P. Beckett et al., Drug Discov. Today 1 (1996) 16-26]. The opposite applies to right-hand-side inhibitors. phosphonate inhibitor of MMP-8. Its uncommon mode of binding to MMP-8 was mainly ascribed to the presence of the proline residue in P(3). Ten new analogues of 2b were obtained by replacement of the aminoterminal l-Pro with aminoacid residues bearing small side chains. Most of the new analogues show an increase of affinity for MMP-2 and MMP-8, and different profiles of selectivity. Computer simulations were performed to explain the effects of substitutions on the preferred mode of binding. They reveal that most of the new analogues are probably accommodated in the right, rather than left-hand side of MMP-8 active site.