Perfluorinated Probes for Noncovalent Protein Recognition and Isolation.

Perfluorinated organic compounds (PFCs) are nontoxic, biocompatible, bioavailable, and bioorthogonal species which possess the unique ability to segregate away from both polar and nonpolar solvents producing a compact fluorophilic phase. Traditional techniques of fluorous chemical proteomics are generally applied to enrich biological samples in target protein(s) exploiting this property of PFCs to build fluorinated probes able to covalently bind to protein ensembles and being selectively extracted by fluorophilic solvents. Aiming at building a strategy able to avoid irreversible modification of the analyzed biosystem, a novel fully noncovalent probe is presented as an enabling tool for the recognition and isolation of biological protein(s). In our strategy, both the fluorophilic extraction and the biorecognition of a selected protein successfully occur via the establishment of reversible but selective interactions.

Synthesis and antimycobacterial activity of (+)-usnic acid conjugates.

New therapeutics are urgently needed to fight tuberculosis and mycobacteria-related diseases that are a major health hazard especially in poor countries. Natural products have been the source of important antitubercular drugs in the past and still need to receive attention as a potent reservoir of chemical structures. Fifteen known and two new (+)-usnic acid (a benzofurandione formerly isolated from lichens) enamines and hydrazones are here described and tested against sensitive and multidrug-resistant strains of mycobacteria. Among several (+)-usnic acid conjugates, PS14 and PS18 showed potent activity against both susceptible and resistant Mycobacterium tuberculosis strains (MIC values of 1-32 and 2-32 mg/L, respectively) comparable with MIC of other antitubercular drugs already in use for tuberculosis treatment.

In Vivo and In Vitro Activities and ADME-Tox Profile of a Quinolizidine-Modified 4-Aminoquinoline: A Potent Anti-P. falciparum and Anti-P. vivax Blood-Stage Antimalarial.

Natural products are a prolific source for the identification of new biologically active compounds. In the present work, we studied the in vitro and in vivo antimalarial efficacy and ADME-Tox profile of a molecular hybrid (AM1) between 4-aminoquinoline and a quinolizidine moiety derived from lupinine (Lupinus luteus). The aim was to find a compound endowed with the target product profile-1 (TCP-1: molecules that clear asexual blood-stage parasitaemia), proposed by the Medicine for Malaria Venture to accomplish the goal of malaria elimination/eradication. AM1 displayed a very attractive profile in terms of both in vitro and in vivo activity. By using standard in vitro antimalarial assays, AM1 showed low nanomolar inhibitory activity against chloroquine-sensitive and resistant P. falciparum strains (range IC50 16-53 nM), matched with a high potency against P. vivax field isolates (Mean IC50 29 nM). Low toxicity and additivity with artemisinin derivatives were also demonstrated in vitro. High in vivo oral efficacy was observed in both P.berghei and P. yoelii mouse models with IC50 values comparable or better than those of chloroquine. The metabolic stability in different species and the pharmacokinetic profile in the mouse model makes AM1 a compound worth further investigation as a potential novel schizonticidal agent.

Theranostic Nanocages for Imaging and Photothermal Therapy of Prostate Cancer Cells by Active Targeting of Neuropeptide-Y Receptor.

Gold nanocages (AuNCs) have been shown to be a useful tool for harnessing imaging and hyperthermia therapy of cancer, thanks to their unique optical properties, low toxicity, and facile surface functionalization. Herein, we use AuNCs for selective targeting of prostate cancer cells (PC3) via specific interaction between neuropeptide Y (NPY) receptor and three different NPY analogs conjugated to AuNCs. Localized surface plasmon resonance band of the nanoconjugates was set around 800 nm, which is appropriate for in vivo applications. Long-term stability of nanoconjugates in different media was confirmed by UV-vis and DLS studies. Active NPY receptor targeting was observed by confocal microscopy showing time-dependent AuNCs cellular uptake. Activation of ERK1/2 pathway was evaluated by Western blot to confirm the receptor-mediated specific interaction with PC3. Cellular uptake kinetics were compared as a function of peptide structure. Cytotoxicity of nanoconjugates was evaluated by MTS and Annexin V assays, confirming their safety within the concentration range explored. Hyperthermia studies were carried out irradiating the cells, previously incubated with AuNCs, with a pulsed laser at 800 nm wavelength, showing a heating enhancement ranging from 6 to 35 °C above the culture temperature dependent on the irradiation power (between 1.6 and 12.7 W/cm2). Only cells treated with AuNCs underwent morphological alterations in the cytoskeleton structure upon laser irradiation, leading to membrane blebbing and loss of microvilli associated with cell migration. This effect is promising in view of possible inhibition of proliferation and invasion of cancer cells. In summary, our Au-peptide NCs proved to be an efficient theranostic nanosystem for targeted detection and activatable killing of prostate cancer cells.

"Blind" targeting in action: From phage display to breast cancer cell targeting with peptide-gold nanoconjugates.

Tumor homing peptides (THPs) specific for a representative breast cancer cell line (MCF-7) were carefully selected basing on a phage-displayed peptide library freely available on the web, namely the "TumorHoPe: A Database of Tumor Homing Peptides". The selected THPs were synthesized and evaluated in terms of their affinity toward MCF-7 cells. Out of 5 tested THPs, 3 best-performing peptide sequences and 1 scrambled sequence were separately conjugated to spherical gold nanoparticles yielding stable nanoconjugates. THP nanoconjugates were examined for their ability to actively target MCF-7 cells in comparison to noncancerous 3T3-L1 fibroblast cells. These THP-gold nanoconjugates exhibited good selectivity and binding affinity by flow cytometry, and low cytotoxicity as assayed by cell death experiments. The uptake of targeted nanoconjugates by the breast cancer cells was confirmed by transmission electron microscopy analysis. This work demonstrates that it is possible to exploit the conjugation of short peptides selected from phage-displayed libraries to develop nanomaterials reliably endowed with tumor targeting potential irrespective of a specific knowledge of the target cell biology.

Synthetic peptides derived from the C-terminal 6kDa region of Plasmodium falciparum SERA5 inhibit the enzyme activity and malaria parasite development.

BACKGROUND: Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site. METHODS: In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential. RESULTS: The final proteolytic step of PfSERA5 involves removal of a C-terminal ~6kDa fragment that results in the generation of a catalytically active ~50kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~6kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5. CONCLUSIONS: Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress. GENERAL SIGNIFICANCE: These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~6kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.

Favorable Preclinical Pharmacological Profile of a Novel Antimalarial Pyrrolizidinylmethyl Derivative of 4-amino-7-chloroquinoline with Potent In Vitro and In Vivo Activities.

The 4-aminoquinoline drugs, such as chloroquine (CQ), amodiaquine or piperaquine, are still commonly used for malaria treatment, either alone (CQ) or in combination with artemisinin derivatives. We previously described the excellent in vitro activity of a novel pyrrolizidinylmethyl derivative of 4-amino-7-chloroquinoline, named MG3, against P. falciparum drug-resistant parasites. Here, we report the optimized and safer synthesis of MG3, now suitable for a scale-up, and its additional in vitro and in vivo characterization. MG3 is active against a panel of P. vivax and P. falciparum field isolates, either alone or in combination with artemisinin derivatives. In vivo MG3 is orally active in the P. berghei, P. chabaudi, and P. yoelii models of rodent malaria with efficacy comparable, or better, than that of CQ and of other quinolines under development. The in vivo and in vitro ADME-Tox studies indicate that MG3 possesses a very good pre-clinical developability profile associated with an excellent oral bioavailability, and low toxicity in non-formal preclinical studies on rats, dogs, and non-human primates (NHP). In conclusion, the pharmacological profile of MG3 is in line with those obtained with CQ or the other quinolines in use and seems to possess all the requirements for a developmental candidate.

Antiplasmodial activities of 4-aminoquinoline-statine compounds.

We report the discovery of new potent inhibitors of the growth of Plasmodium falciparum chloroquine (CQ)-resistant W2 strain. These compounds were designed using the double drug approach by introducing a residue able to enhance the accumulation of plasmepsins inhibitors into the food vacuole. Some of the molecules were more active than CQ against CQ-resistant strain and showed good selectivity against cathepsin D.

Synthesis and comparison of antiplasmodial activity of (+), (-) and racemic 7-chloro-4-(N-lupinyl)aminoquinoline.

Recently the N-(-)-lupinyl-derivative of 7-chloro-4-aminoquinoline ((-)-AM-1; 7-chloro-4-{N-[(1S,9aR)(octahydro-2H-quinolizin-1-yl)methyl]amino}quinoline) showed potent in vitro and in vivo activity against both Chloroquine susceptible and resistant strains of Plasmodium falciparum. However, (-)-AM-1 is synthesized starting from (-)-lupinine, an expensive alkaloid isolated from Lupinus luteus whose worldwide production is not sufficient, at present, for large market purposes. To overcome this issue, the corresponding racemic compound, derived from synthetic (±)-lupinine was considered a cheaper alternative for the development of a novel antimalarial agent. Therefore, the racemic and the 7-chloro-4-(N-(+)-lupinyl)aminoquinoline ((±)-AM-1; (+)-AM-1) were synthesized and their in vitro antimalarial activity and cytotoxicity compared with those of (-)-AM-1. The (+)-lupinine required for the synthesis of (+)-AM-1 was obtained through a not previously described lipase catalyzed kinetic resolution of (±)-lupinine. In terms of antimalarial activity, (±)-AM1 and (+)-AM1 demonstrated very good activity in vitro against both CQ-R and CQ-S strains of P. falciparum (range IC(50) 16-35 nM), and low toxicity against human normal cell lines (therapeutic index >1000), comparable with that of (-)-AM1. These results confirm that the racemate (±)-AM1 could be considered as a potential antimalarial agent, ensuring a decrease of costs of synthesis compared to (-)-AM1.

From DC18 to MR07: A Metabolically Stable 4,4'-Oxybisbenzoyl Amide as a Low-Nanomolar Growth Inhibitor of P. falciparum.

To improve the metabolic stability of a 4,4'-oxybisbenzoyl-based novel and potent (nanomolar-range IC50 ) antiplasmodial agent previously described by us, in silico-guided structure-activity relationship (SAR) campaigns have been conducted to substitute its peptide decorations with more metabolically stable residues. The effects of the various structural modifications were then correlated with the antiplasmodial activity in vitro in phenotypic assays. Among the several derivatives synthetized and compared with the 3D-pharmacophoric map of the original lead, a novel compound, characterized by a western tert-butyl glycine residue and an eastern 1S,2S-aminoacyclohexanol, showed low-nanomolar-range antiplasmodial activity, no signs of cross-resistance and, most importantly, 47-fold improved Phase I metabolic stability when incubated with human liver microsomes. These results highlight the efficacy of in silico-guided SAR campaigns which will allow us to further optimize the structure of the new lead aiming at testing its efficacy in vivo using different routes of administration.

Novel biotinylated bile acid amphiphiles: micellar aggregates formation and interaction with hepatocytes.

Amphiphilic bile acids linked through an oligoethylene glycol to a biotin moiety were synthesized and shown to create micellar structures in aqueous environment, interact with avidin and be efficiently incorporated into hepatocyte cells, suggesting their potential as a drug delivery system against liver diseases.

Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria.

BACKGROUND: The sun-dried rind of the immature fruit of pomegranate (Punica granatum) is presently used as a herbal formulation (OMARIA, Orissa Malaria Research Indigenous Attempt) in Orissa, India, for the therapy and prophylaxis of malaria. The pathogenesis of cerebral malaria, a complication of the infection by Plasmodium falciparum, is an inflammatory cytokine-driven disease associated to an up-regulation and activity of metalloproteinase-9 and to the increase of TNF production. The in vitro anti-plasmodial activity of Punica granatum (Pg) was recently described. The aim of the present study was to explore whether the anti-malarial effect of OMARIA could also be sustained via other mechanisms among those associated to the host immune response. METHODS: From the methanolic extract of the fruit rind, a fraction enriched in tannins (Pg-FET) was prepared. MMP-9 secretion and expression were evaluated in THP-1 cells stimulated with haemozoin or TNF. The assays were conducted in the presence of the Pg-FET and its chemical constituents ellagic acid and punicalagin. The effect of urolithins, the ellagitannin metabolites formed by human intestinal microflora, was also investigated. RESULTS: Pg-FET and its constituents inhibited the secretion of MMP-9 induced by haemozoin or TNF. The effect occurred at transcriptional level since MMP-9 mRNA levels were lower in the presence of the tested compounds. Urolithins as well inhibited MMP-9 secretion and expression. Pg-FET and pure compounds also inhibited MMP-9 promoter activity and NF-kB-driven transcription. CONCLUSIONS: The beneficial effect of the fruit rind of Punica granatum for the treatment of malarial disease may be attributed to the anti-parasitic activity and the inhibition of the pro-inflammatory mechanisms involved in the onset of cerebral malaria.

Design, synthesis and docking studies of Hydroxyethylamine and Hydroxyethylsulfide BACE-1 inhibitors.

Both stereoisomer of hydroxyethylamine (HEA) and hydroxyethylsulfide (HES) transition-state isostere inhibitors of BACE-1 were synthesized. The syn-HEA epimer resulted always more active than the anti stereoisomer independently from the P(1) and the P(1)' substituents. On the contrary, the anti epimer of the HES isostere resulted more active than the syn stereoisomer. The change of stereopreference was studied by molecular modelling.

Discovery and Pharmacophore Mapping of a Low-Nanomolar Inhibitor of P. falciparum Growth.

The treatment of malaria, the most common parasitic disease worldwide and the third deadliest infection after HIV and tuberculosis, is currently compromised by the dramatic increase and diffusion of drug resistance among the various species of Plasmodium, especially P. falciparum (Pf). In this view, the development of new antiplasmodial agents that are able to act via innovative mechanisms of action, is crucial to ensure efficacious antimalarial treatments. In one of our previous communications, we described a novel class of compounds endowed with high antiplasmodial activity, characterized by a pharmacophore never described before as antiplasmodial and identified by their 4,4'-oxybisbenzoyl amide cores. Here, through a detailed structure-activity relationship (SAR) study, we thoroughly investigated the chemical features of the reported scaffolds and successfully built a novel antiplasmodial agent active on both chloroquine (CQ)-sensitive and CQ-resistant Pf strains in the low nanomolar range, without displaying cross-resistance. Moreover, we conducted an in silico pharmacophore mapping.

Parallel synthesis and antileishmanial activity of ether-linked phospholipids.

The synthesis and antileishmanial activity of 18 edelfosine analogues are described. Compounds were obtained in parallel combining solid phase and solution phase synthesis. The most active analogue is characterized by the octadecyl group in position 2 of the glycerol chain. Considering that this substitution determines the loss of antitumor activity, a different mechanism of antileishmanial action can be hypothesized.

Novel amodiaquine congeners as potent antimalarial agents.

To develop new classes of antimalarial agents, the possibility of replacing the phenolic ring of amodiaquine, tebuquine, and isoquine with other aromatic nuclei was investigated. Within a first set of pyrrole analogues, several compounds displayed high activity against both D10 (CQ-S) and W-2 (CQ-R) strains of Plasmodium falciparum. The isoquine structure was also modified by replacing the diethylamino group with more metabolically stable bicyclic moieties and by replacing the aromatic hydroxyl function with a chlorine atom. Among these compounds, two quinolizidinylmethylamino derivatives (6f and 7f) displayed high activity against both CQ-S and CQ-R strains.

Targeting the plasmepsin 4 orthologs of Plasmodium sp. with "double drug" inhibitors.

Plasmepsin 4 (PM4) is a digestive vacuole enzyme found in all Plasmodium species examined to date. While P. falciparum has three additional aspartic proteinases in its digestive vacuole in addition to plasmepsin 4, other Plasmodium species have only PM4 in their digestive vacuole. Therefore, PM4 may be a good target for the development of an antimalarial drug. This study presents data obtained with PM4s from several Plasmodium species. Low nanomolar K(i) values have been observed for all PM4s studied.

High antiplasmodial activity of novel plasmepsins I and II inhibitors.

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the "double-drug" approach: primaquine, which has been linked to statine-based inhibitors of plasmepsins (PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC(50)) as low as 0.1 microM was obtained, an excellent improvement in comparison with inhibitors previously reported (IC(50) = 2-20 microM). The killing activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar range (K(i) = 1-700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human fibroblasts at 100 microM and were highly selective for PLMs vs human cathepsin D.

[4-(2H-1,2,3-benzotriazol-2-yl)phenoxy]alkanoic acids as agonists of peroxisome proliferator-activated receptors (PPARs).

A series of [4-(2H-1,2,3-benzotriazol-2-yl)phenoxy]alkanoic acids has been synthesized and tested as agonists of Peroxisome Proliferator-Activated Receptor (PPAR) alpha, gamma, and delta. Three compounds displayed 56 to 96% of maximal activity of the reference drug Wy-14643 on PPARalpha, and two of these, i.e., 1 and 5, exhibited also moderate activity on either PPARgamma or delta with efficacy equal to 50% and 46% of that of rosiglitazone and GW 501516, respectively. Thus, compounds 1 and 5 represent interesting starting point for preparing novel agents for the treatment of dyslipidemia or of dyslipidemic type-2 diabetes.

Picomolar Inhibition of Plasmepsin V, an Essential Malaria Protease, Achieved Exploiting the Prime Region.

Malaria is an infectious disease caused by Plasmodium parasites. It results in an annual death-toll of ~ 600,000. Resistance to all medications currently in use exists, and novel antimalarial drugs are urgently needed. Plasmepsin V (PmV) is an essential Plasmodium protease and a highly promising antimalarial target, which still lacks molecular characterization and drug-like inhibitors. PmV, cleaving the PExEl motif, is the key enzyme for PExEl-secretion, an indispensable parasitic process for virulence and infection. Here, we describe the accessibility of PmV catalytic pockets to inhibitors and propose a novel strategy for PmV inhibition. We also provide molecular and structural data suitable for future drug development. Using high-throughput platforms, we identified a novel scaffold that interferes with PmV in-vitro at picomolar ranges (~ 1,000-fold more active than available compounds). Via systematic replacement of P and P' regions, we assayed the physico-chemical requirements for PmV inhibition, achieving an unprecedented IC50 of ~20 pM. The hydroxyethylamine moiety, the hydrogen acceptor group in P2', the lipophilic groups upstream to P3, the arginine and other possible substitutions in position P3 proved to be critically important elements in achieving potent inhibition. In-silico analyses provided essential QSAR information and model validation. Our inhibitors act 'on-target', confirmed by cellular interference of PmV function and biochemical interaction with inhibitors. Our inhibitors are poorly performing against parasite growth, possibly due to poor stability of their peptidic component and trans-membrane permeability. The lowest IC50 for parasite growth inhibition was ~ 15 µM. Analysis of inhibitor internalization revealed important pharmacokinetic features for PExEl-based molecules. Our work disclosed novel pursuable drug design strategies for highly efficient PmV inhibition highlighting novel molecular elements necessary for picomolar activity against PmV. All the presented data are discussed in respect to human aspartic proteases and previously reported inhibitors, highlighting differences and proposing new strategies for drug development.