Identification of West Nile virus RNA-dependent RNA polymerase non-nucleoside inhibitors by real-time high throughput fluorescence screening.

West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 µM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1-4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 µM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors.

Identification of L. infantum trypanothione synthetase inhibitors with leishmanicidal activity from a (non-biased) in-house chemical library.

Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypanothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 µM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 µM and a selectivity index of 35.

Antinociceptive and modulatory effect of pathoplastic changes in spinal glia of a TLR4/CD14 blocking molecule in two models of pain in rat.

The role of spinal glia in the development and maintenance of chronic pain has become over the last years a subject of increasing interest. In this regard, toll-like receptor 4 (TLR4) signaling has been proposed as a major trigger mechanism. Hence, in this study we explored the implications of TLR4 inhibition in the periphery and primarily in the CNS, focusing on the impact this inhibition renders in pain development and glia activation in the dorsal horn in two models of pain. Making use of a synthetic cluster of differentiation 14 (CD14)/TLR4 antagonist, the effect of TLR4 blockade on tactile allodynia and heat hyperalgesia was evaluated in osteoarthritic and postoperative rat models. An in vitro parallel artificial membrane permeation assay was performed to determine the proneness of the drug to permeate the blood-brain barrier prior to systemic and central administration. Findings suggest a dominant role of peripheral TLR4 in the model of incisional pain, whilst both peripheral and central TLR4 seem to be responsible for osteoarthritic pain. That is, central and peripheral TLR4 may be differently involved in the etiopathology of diverse types of pain what potentially seems a promising approach in the management of pain.

TLR4 Antagonism Reduces Movement-Induced Nociception and ATF-3 Expression in Experimental Osteoarthritis.

INTRODUCTION: Toll-like receptor 4 (TLR4) is a pattern recognition receptor involved in the detection of pathogen-associated molecular patterns (PAMPs), but also a "danger-sensing" receptor that recognizes host-derived endogenous molecules called damage-associated molecular patterns (DAMPs). The involvement of TLR4 in rheumatic diseases is becoming evident, as well as its potential role as a target for therapeutic intervention. Moreover, increasing evidence also suggests that TLR4 is implicated in chronic pain states. Thus, in this study, we evaluated whether a systemic administration of a synthetic antagonist of TLR4 (TLR4-A1) could decrease nociception and cartilage degradation in experimental osteoarthritis (OA). Furthermore, as the activation transcription factor (ATF)-3 serves as a negative regulator for TLR4-stimulated inflammatory response, we also evaluated the effect of TLR4 inhibition on ATF-3 expression in primary afferent neurons at the dorsal root ganglia (DRG). METHODS: OA was induced in adult male Wistar rats through an intra-articular injection of 2 mg of sodium mono-iodoacetate (MIA) into the left knee. From days 14 to 28 after OA induction, animals received an intraperitoneal injection of either TLR4-A1 (10 mg/kg) or vehicle. Movement- and loading-induced nociception was evaluated in all animals, by the Knee-Bend and CatWalk tests, before and at several time-points after TLR4-A1/vehicle administration. Immunofluorescence for TLR4 and ATF-3 was performed in L3-L5 DRG. Knee joints were processed for histopathological evaluation. RESULTS: Administration of TLR4-A1 markedly reduced movement-induced nociception in OA animals, particularly in the Knee-Bend test. Moreover, the increase of ATF-3 expression observed in DRG of OA animals was significantly reduced by TLR4-A1. However, no effect was observed in cartilage loss nor in the neuronal cytoplasmic expression of TLR4 upon antagonist administration. CONCLUSION: The TLR4 antagonist administration possibly interrupts the TLR4 signalling cascade, thus decreasing the neurotoxic environment at the joint, which leads to a reduction in ATF-3 expression and in nociception associated with experimental OA.

Double Arylation of the Indole Side Chain of Tri- and Tetrapodal Tryptophan Derivatives Renders Highly Potent HIV-1 and EV-A71 Entry Inhibitors†.

We have recently described a new generation of potent human immunodeficiency virus (HIV) and EV-A71 entry inhibitors. The prototypes contain three or four tryptophan (Trp) residues bearing an isophthalic acid moiety at the C2 position of each side-chain indole ring. This work is now extended by both shifting the position of the isophthalic acid to C7 and synthesizing doubly arylated C2/C7 derivatives. The most potent derivative (50% effective concentration (EC50) HIV-1, 6 nM; EC50 EV-A71, 40 nM), 33 (AL-518), is a C2/C7 doubly arylated tetrapodal compound. Its superior anti-HIV potency with respect to the previous C2-arylated prototype is in consonance with its higher affinity for the viral gp120. 33 (AL-518) showed comparable antiviral activities against X4 and R5 HIV-1 strains and seems to interact with the tip and base of the gp120 V3 loop. Taken together, these findings support the interest in 33 (AL-518) as a useful new prototype for anti-HIV/EV71 drug development.

Scaffold Simplification Strategy Leads to a Novel Generation of Dual Human Immunodeficiency Virus and Enterovirus-A71 Entry Inhibitors.

Currently, there are only three FDA-approved drugs that inhibit human immunodeficiency virus (HIV) entry-fusion into host cells. The situation is even worse for enterovirus EV71 infection for which no antiviral therapies are available. We describe here the discovery of potent entry dual inhibitors of HIV and EV71. These compounds contain in their structure three or four tryptophan (Trp) residues linked to a central scaffold. Critical for anti-HIV/EV71 activity is the presence of extra phenyl rings, bearing one or two carboxylates, at the C2 position of the indole ring of each Trp residue. The most potent derivatives, 22 and 30, inhibit early steps of the replicative cycles of HIV-1 and EV-A71 by interacting with their respective viral surfaces (glycoprotein gp120 of HIV and the fivefold axis of the EV-A71 capsid). The high potency, low toxicity, facile chemical synthesis, and great opportunities for chemical optimization make them useful prototypes for future medicinal chemistry studies.

Conformational mimetics of the α-methyl chalcone TUB091 binding tubulin: Design, synthesis and antiproliferative activity.

Based on the conformation of the α-methyl chalcone TUB091 in its complex with tubulin, a series of conformational mimetics have been designed and synthesized where the methyl group of the chalcone has been fused to phenyl ring B resulting in 1,2,3,4-tetrahydronaphthalen-2-yl aryl ketones. Among the synthesized compounds, the 5-amino-6-methoxy derivative, with a similar substitution pattern to that of TUB091, showed antiproliferative activity around 20 nM against tumor and endothelial cells. Tubulin binding experiments confirmed its binding to tubulin at the colchicine site with a Kb of 2.4 × 106 M-1 resulting in the inhibition of the in vitro assembly of purified tubulin. Moreover, based on the recently reported complex of combretastatin A4 (CA4) with tubulin, a comparative analysis of the binding mode of CA4 and the α-methyl chalcone to tubulin has been performed.

RNase II regulates RNase PH and is essential for cell survival during starvation and stationary phase.

RNase II is the most active exoribonuclease in Escherichia coli cell extracts. Yet, its removal appears to have no deleterious effect on growing cells. Here, we show that RNase II is required for cell survival during prolonged stationary phase and upon starvation. The absence of RNase II leads to greatly increased rRNA degradation and to the accumulation of rRNA fragments, both of which lead to a decline in cell survival. The deleterious effects of RNase II removal can be completely reversed by the simultaneous absence of a second exoribonuclease, RNase PH, an enzyme known to be required to initiate ribosome degradation in starving cells. We have now found that the role of RNase II in this process is to regulate the amount of RNase PH present in starving cells, and it does so at the level of RNase PH stability. RNase PH normally decreases as much as 90% during starvation because the protein is unstable under these conditions; however, in the absence of RNase II the amount of RNase PH remains relatively unchanged. Based on these observations, we propose that in the presence of RNase II, nutrient deprivation leads to a dramatic reduction in the amount of RNase PH, thereby limiting the extent of rRNA degradation and ensuring cell survival during this stress. In the absence of RNase II, RNase PH levels remain high, leading to excessive ribosome loss and ultimately to cell death. These findings provide another example of RNase regulation in response to environmental stress.

Structure-activity relationship studies on a Trp dendrimer with dual activities against HIV and enterovirus A71. Modifications on the amino acid.

We have recently described a new class of dendrimers with tryptophan (Trp) on the surface that show dual antiviral activities against HIV and EV71 enterovirus. The prototype compound of this family is a pentaerythritol derivative with 12 Trps on the periphery. Here we complete the structure-activity relationship studies of this family to identify key features that might be significant for the antiviral activity. With this aim, novel dendrimers containing different amino acids (aromatic and non-aromatic), tryptamine (a "decarboxylated" analogue of Trp) and N-methyl Trp on the periphery have been prepared. Dendrimer with N-Methyl Trp was the most active against HIV-1 and HIV-2 while dendrimer with tyrosine was endowed with the most potent antiviral activity against EV71. This tyrosine dendrimer proved to inhibit a large panel of EV71 clinical isolates (belonging to different clusters) in the low nanomolar/high picomolar range. In addition, a new synthetic procedure (convergent approach) has been developed for the synthesis of the prototype and some other dendrimers. This convergent approach proved more efficient (higher yields, easier purification) than the divergent approach previously reported.

Optimization of a Class of Tryptophan Dendrimers That Inhibit HIV Replication Leads to a Selective, Specific, and Low-Nanomolar Inhibitor of Clinical Isolates of Enterovirus A71.

Tryptophan dendrimers that inhibit HIV replication by binding to the HIV envelope glycoproteins gp120 and gp41 have unexpectedly also proven to be potent, specific, and selective inhibitors of the replication of the unrelated enterovirus A71. Dendrimer 12, a consensus compound that was synthesized on the basis of the structure-activity relationship analysis of this series, is 3-fold more potent against the BrCr lab strain and, surprisingly, inhibits a large panel of clinical isolates in the low-nanomolar/high-picomolar range.

Anti-HIV-1 activity of a tripodal receptor that recognizes mannose oligomers.

The glycoprotein gp120 of the HIV-1 viral envelope has a high content in mannose residues, particularly α-1,2-mannose oligomers. Compounds that interact with these high-mannose type glycans may disturb the interaction between gp120 and its (co)receptors and are considered potential anti-HIV agents. Previously, we demonstrated that a tripodal receptor (1), with a central scaffold of 1,3,5-triethylbenzene substituted with three 2,3,4-trihydroxybenzoyl groups, selectively recognizes α-1,2-mannose polysaccharides. Here we present additional studies to determine the anti-HIV-1 activity and the mechanism of antiviral activity of this compound. Our studies indicate that 1 shows anti-HIV-1 activity in the low micromolar range and has pronounced gp120 binding and HIV-1 integrase inhibitory capacity. However, gp120 binding rather than integrase inhibition seems to be the primary mechanism of antiviral activity of 1.

Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41.

Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.

Linear and branched alkyl-esters and amides of gallic acid and other (mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-HCV inhibitors.

Linear and branched compounds that contain two, three or five units of galloyl (3,4,5-trihydroxybenzoyl) or its isomer 2,3,4-trihydroxybenzoyl, as well as other mono- or dihydroxybenzoyl moieties have been synthesized. These molecules have been evaluated for their in vitro inhibitory effects against a wide panel of viruses showing preferential activity against HIV and HCV. Our structure-activity relationship studies demonstrated that the 2,3,4-trihydroxybenzoyl moiety provides better antiviral activities than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins. This observation can be of interest for the further rational exploration of compounds with anti-HCV/HIV properties. The most notable finding with respect to HIV is that the tripodal compounds 43 and 45, with three 2,3,4-trihydroxybenzoyl moieties, showed higher activities than linear compounds with only one or two. With respect to HCV, the linear compounds, 52 and 41, containing a 12 polymethylene chain and two 2,3 di- or 2,3,4 tri-hydroxybenzoyl groups respectively at the ends of the molecule showed good antiviral efficiency. Furthermore, the anti-HCV activity of both compounds was observed at concentrations well below the cytotoxicity threshold. A representative member of these compounds, 41, showed that the anti-HCV activity was largely independent of the genetic make-up of the HCV subgenomic replicon and cell lines used.

Multivalent agents containing 1-substituted 2,3,4-trihydroxyphenyl moieties as novel synthetic polyphenols directed against HIV-1.

The synthesis and the assessment of the anti-HIV activity of a set of molecules inspired by the multivalent structures of some naturally-occurring polyphenols (tannins) are reported. Different multibranched scaffolds have been derived from pentaerythritol as the central core which distribute spatially synthetic polyphenolic subunits based on 1-substituted 2,3,4-trihydroxyphenyl moieties. A tetrapodal compound () bearing four N-(2,3,4-trihydroxyphenyl)amide groups, exhibits remarkable selective activity against HIV-1 with EC50 values in the micromolar scale, in the same range as those reported for the most representative anti-HIV tannins. Preliminary SAR studies emphasize the importance of the 1-substituted 2,3,4-trihydroxyphenyl moiety, the presence of an amide as the linker and the multivalent architecture of these molecules, since the anti-HIV activity increases with the number of polyphenolic moieties. The data support the interest in synthetic polyphenols and represent a promising starting point for further design and development of selective HIV-1 inhibitors.

Selective inhibition of Human Immunodeficiency Virus type 1 (HIV-1) by a novel family of tricyclic nucleosides.

Nucleoside 1, with an unusual tricyclic carbohydrate moiety, specifically inhibits HIV-1 replication while being inactive against HIV-2 or other (retro) viruses. In an attempt to increase the inhibitory efficacy against HIV-1, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on this prototype compound. These include substitution of the ethoxy group at the C-4″ position by alkoxy groups of different length, branching, conformational freedom or functionalization. In addition, the 4″-ethoxy group has been removed or substituted by other functional groups. The role of the tert-butyldimethylsilyl (TBDMS) group at the 2' position has also been studied by preparing the corresponding 2'-deprotected derivative or by replacing it by other silyl (tert-hexyldimethylsilyl) or acyl (acetyl) moieties. Finally, the thymine of the prototype compound has been replaced by N-3-methylthymine, uracil or thiophenyl. Some of these compounds were endowed with a 6- to 7-fold higher selectivity than the prototype 1. The tricyclic nucleosides here described represent a novel type of selective anti HIV-1 inhibitors, targeted at the HIV-1-encoded reverse transcriptase.

One-pot synthesis of polycyclic nucleosides with unusual molecular skeletons.

An alpha hydroxy pyrrolidine tricyclic nucleoside 3 and its spontaneous reaction with acetone is described. In this transformation highly functionalized polycyclic nucleosides with rather unusual molecular skeletons are formed in a complete regio- and stereoselective way. The reaction involves the formation of three new bonds, two of them novel carbon-carbon bonds, in a one-pot way. An enamine-iminium mechanism with participation of carbinolamine, iminium ion, and enamine intermediates is proposed as a plausible explanation for this transformation. The scope of the reaction is briefly studied concluding that the nature of the ketone (R(1)COR(2)) is critical for the initial attack of the NH to the carbonyl group.

Novel N-3 substituted TSAO-T derivatives: synthesis and anti-HIV-evaluation.

Novel derivatives of the anti-HIV-1 agent, TSAO-T, bearing at the N-3 position alkylating groups or photoaffinity labels were prepared and evaluated for their anti-HIV activity. All of these compounds demonstrated pronounced anti-HIV-1 activity and inhibited HIV-1 RT; however, we were unable to detect stable covalent linkages between inhibitor and enzyme. In addition, compounds with an alcohol functional group connected to the N-3 position through a cis or trans double bond have been prepared. These compounds have been useful to study how the conformational restriction of the linker affects in the interaction between the N-3 substituent and the HIV-1 RT enzyme.

Synthesis of highly condensed polycyclic carbohydrates by reaction of a spirocyclic enamino sulfonate derived from d-xylofuranose with bifunctional reagents.

The appropriately substituted 5-O-tosyl derivative (1), easily prepared from 1,2-O-isopropylidene-alpha-d-xylofuranose, serves as a useful precursor for the preparation of highly condensed cyclic carbohydrates. The synthesis involves a first cyclization of the 5-O-tosyl sugar derivative 1 to a highly reactive cyclic enamine, which subsequently undergoes the nucleophilic attack of a bifunctional reagent X(CH2)nZ in a regio- and stereospecific way. Finally, a spontaneous cyclization step allows the formation of a stereochemically defined extra ring, fused to the sugar backbone. The functionalization and size of this ring can be varied by the proper choice of the bifunctional reagent. X-ray diffraction analysis and intensive NMR studies with one of these carbohydrates were performed to highlight the strained nature of these compounds.

A cyclic enamine derived from 1,2-O-isopropylidene-alpha-D-xylofuranose as a novel carbohydrate intermediate to achieve skeletal diversity.

The commercially available carbohydrate 1,2-O-isopropylidene-alpha-D-xylofuranose was efficiently transformed into the high-added-value synthetic scaffold 8. The transformation requires the synthesis of the 5-O-tosyl derivative 7 and its subsequent intramolecular cyclization under basic conditions to give the cyclic enamine 8. Reaction of 8 with O-, N-, S-, and C-nucleophiles and amino acids allowed its efficient transformation (one-step, high yields, and easy purifications) into fused cyclic sugar derivatives with rather unusual molecular skeletons in a completely regio- and stereoselective manner. The characteristics of the sugar derivative 8 established here, high reactivity, synthetic accessibility, and the potential for conversion into a vast collection of products by the action of different nucleophiles, indicate that it will prove to be a useful chiral intermediate for achieving skeletal diversity. The constrained structures and dense functionalization of the polycyclic sugar derivatives generated from 8 make these compounds promising candidates for use as starting agents for the production of new analogues and as drugs.

Dynamics of bolaamphiphilic fluorescent polyenes in lipid bilayers from polarization emission spectroscopy.

The rotational motions of the biamphiphilic polyenes (bolapolyenes) dimethyl all-(E)-octacosa-10,12,14,16,18-pentaenedioate (DE28:5) and dimethyl all-(E)-tetratriaconta-13,15,17,19,21-pentaenedioate (DE34:5), with head-to-head distances of 34 and 42A, respectively, have been examined by fluorescence anisotropy methods. The membrane-spanning bolapolyenes, which contain a central emitting pentaene group tethered to two methoxycarbonyl opposite polar heads by symmetric C(8) (DE28:5) and C(11) (DE34:5) polymethylene chains, were dispersed in lipid bilayers of DPPC or DMPC, and the stationary and picosecond-resolved emission was recorded as a function of temperature. In fluid-phase DMPC bilayers, three relaxation times could be determined, assigned to fast (0.2 and 2ns) single-bond isomerization processes localized on the alkyl chains, and to whole-molecule oscillations ( approximately 11ns), respectively. The anisotropy decay parameters were further analyzed in terms of a diffusive model for wobbling in a Gaussian ordering potential, to assess the anchoring effect of the symmetric polar heads. In this way, the average rotational diffusion constant of the bolapolyenes, D( perpendicular), could be estimated as 0.022-0.026rad(2) ns(-1) (DMPC bilayers, 35 degrees Celsius), a value that is only 1/3 of that corresponding to the related pentaene fatty acid spanning a single membrane monolayer. In contrast, the amplitude of the equilibrium orientational distribution (theta(half-cone) approximately 50 degrees ) is very similar for both the transmembrane and the single-headed polyenes. The reorientational oscillations of the central emitting group in the bolapolyenes necessarily would produce large-amplitude (2-5A) and very fast (ns) translational motions of the polar heads.