Selective inhibition of HIV-1 reverse transcriptase (HIV-1 RT) RNase H by small RNA hairpins and dumbbells

We present here the design of a novel class of RNA inhibitors of the RNase H domain of HIV-1 RT, a ribonuclease activity that is essential for viral replication in vivo. Specifically, we show that small RNA hairpins and dumbbells can selectively inhibit the RNase H activity of HIV-1 RT without affecting other cellular RNases H (e.g., E. coli and human RNase H). These results suggest that the inhibitors do not interact with the nucleic acid binding site of RT RNase H, as this region should be well conserved among the various enzymes. The most potent inhibitors displayed IC50 values in the 3-8 microM range. Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by the hairpin and dumbbell aptamers, a property not previously observed for any nucleic acid aptamer directed against RT RNase H. The results described here suggest a noncompetitive binding mechanism, as outlined in the differential inhibitory characteristics of each of the nucleic acid aptamers against the bacterial, human, and viral RNase H homologues.     

Design, synthesis, biological evaluation, and molecular modeling studies of TIBO-like cyclic sulfones as non-nucleoside HIV-1 reverse transcriptase inhibitors

TIBO- and TBO-like sulfone derivatives 1 and 2 were designed, synthesized, and tested for their ability to block the replication cycle of HIV-1 in infected cells. The anti-HIV-1 activities of sulfones 3, which were intermediates in the syntheses of 1 and 2, were also evaluated. Surprisingly, the sulfone analogues of TIBO R82913 (compounds 1) were inactive, whereas interesting results were obtained for truncated derivatives 2. Compound 2 w was the most potent among this series in cell-based assays (EC50=0.07 microM, CC50>200 microM, SI>2857). It was twofold less potent than R82913, but more selective. An X-ray crystallographic analysis was carried out to establish the absolute configuration of 2 w and its enantiomer 2 x, which were obtained by semipreparative HPLC of 2 v, one of the most potent racemates. Compounds 1-3 were proven to target HIV-1 RT. In fact, representative derivatives inhibited recombinant HIV-1 RT in vitro at concentrations similar to those active in cell-based assays. 3D QSAR studies and docking simulations were developed on TIBO- and TBO-like sulfone derivatives to rationalize their anti-HIV-1 potencies and to predict the activity of novel untested sulfone derivatives. Predictive 3D QSAR models were obtained with a receptor-based alignment by docking of TIBO- and TBO-like derivatives into the NNBS of RT.     

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC₅₀ value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.     

Synthesis,Biological Activity,and Molecular Dynamics Study of Novel Series of a Trimethoprim Analogs as Multi-Targeted Compounds: Dihydrofolate Reductase (DHFR) Inhibitors and DNA-Binding Agents

Eighteen previously undescribed trimethoprim (TMP) analogs containing amide bonds (1–18) were synthesized and compared with TMP, methotrexate (MTX), and netropsin (NT). These compounds were designed as potential minor groove binding agents (MGBAs) and inhibitors of human dihydrofolate reductase (hDHFR). The all-new derivatives were obtained via solid phase synthesis using 4-nitrophenyl Wang resin. Data from the ethidium displacement test confirmed their DNA-binding capacity. Compounds 13–14 (49.89% and 43.85%) and 17–18 (41.68% and 42.99%) showed a higher binding affinity to pBR322 plasmid than NT. The possibility of binding in a minor groove as well as determination of association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)², and poly (dG-dC)². With the exception of compounds 9 (IC50 = 56.05 µM) and 11 (IC50 = 55.32 µM), all of the compounds showed better inhibitory properties against hDHFR than standard, which confirms that the addition of the amide bond into the TMP structures increases affinity towards hDHFR. Derivatives 2, 6, 13, 14, and 16 were found to be the most potent hDHFR inhibitors. This molecular modelling study shows that they interact strongly with a catalytically important residue Glu-30.     

Discovery of N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as HCV NS5B polymerase inhibitors

The metal ion chelating β-N-hydroxy-γ-ketocarboxamide pharmacophore was integrated into a quinazolinone scaffold, leading to N-arylalkyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives as hepatitis C virus (HCV) NS5B polymerase inhibitors. Lead optimization led to the identification of N-phenylpropyl carboxamide 9 k (IC(50) =8.8 μM). Compound 9 k possesses selectivity toward HCV1b replicon Ava.5 cells (EC(50) =17.5 μM) over parent Huh-7 cells (CC(50) =187.5 μM). Compound 9 k effects a mixed mode of NS5B inhibition, with NTP-competitive displacement properties. The interaction between 9 k and NS5B is stabilized by the presence of magnesium ions. Docking studies showed that the binding orientation of 9 k occupies the central portions of both magnesium-mediated and NTP-ribose-response binding sites within the active site region of NS5B. As a result, 3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide derivatives are disclosed herein as novel, mainly active site inhibitors of HCV NS5B polymerase.     

Synthesis and Anticancer Activity of Mitotic-Specific 3,4-Dihydropyridine-2(1H)-thiones

Most anticancer drugs target mitosis as the most crucial and fragile period of rapidly dividing cancer cells. However the limitations of classical chemotherapeutics drive the search for new more effective and selective compounds. For this purpose structural modifications of the previously characterized pyridine analogue (S1) were incorporated aiming to obtain an antimitotic inhibitor of satisfactory and specific anticancer activity. Structure-activity relationship analysis of the compounds against a panel of cancer cell lines allowed to select a compound with a thiophene ring at C5 of a 3,4-dihydropyridine-2(1H)-thione (S22) with promising antiproliferative activity (IC₅₀ equal 1.71 ± 0.58 µM) and selectivity (SI = 21.09) against melanoma A375 cells. Moreover, all three of the most active compounds from the antiproliferative study, namely S1, S19 and S22 showed better selectivity against A375 cells than reference drug, suggesting their possible lower toxicity and wider therapeutic index. As further study revealed, selected compounds inhibited tubulin polymerization via colchicine binding site in dose dependent manner, leading to aberrant mitotic spindle formation, cell cycle arrest and apoptosis. Summarizing, the current study showed that among obtained mitotic-specific inhibitors analogue with thiophene ring showed the highest antiproliferative activity and selectivity against cancer cells.     

Hyrtiosal, from the marine sponge Hyrtios erectus, inhibits HIV-1 integrase binding to viral DNA by a new inhibitor binding site

HIV-1 integrase (IN) is composed of three domains, the N-terminal domain (NTD, residues 1-50), the catalytic core domain (CCD, residues 51-212), and the C-terminal domain (CTD, residues 213-288). All the three domains are required for the two known integration reactions. CCD contains the catalytic triad and is believed to bind viral DNA specifically, and CTD binds viral DNA in a nonspecific manner. As no clear evidence has confirmed the involvement of NTD in DNA binding directly, NTD has not been seriously considered and less is known about its function in viral replication. In the current work, using a SPR technology-based assay, the HIV-1 viral DNA was determined to bind directly to NTD with a K(D) value of 8.8 microM, suggesting that the process of preintegrated complex formation for HIV-1 IN might involve the direct interaction of NTD with viral DNA in addition to binding of viral DNA to the catalytic core domain and C-terminal domain. Moreover, such viral DNA/IN binding could be inhibited by the marine product hyrtiosal from the marine sponge Hyrtios erectus with an IC(50) of 9.60+/-0.86 microM. Molecular dynamic analysis correlated with a site-directed mutagenesis approach further revealed that such hyrtiosal-induced viral DNA/IN binding inhibition was caused by the fact that hyrtiosal could bind HIV-1 NTD at Ser17, Trp19, and Lys34. As hyrtiosal was recently discovered by us as a protein tyrosine phosphatase 1B (PTP1B) inhibitor,1 this work might also supply multiple-target information for this marine product, and the verified HIV-NTD/HIV-1 IN interaction model could have further implications for new HIV-1 IN inhibitor design and evaluation.     

Carborane-Containing Matrix Metalloprotease (MMP) Ligands as Candidates for Boron Neutron-Capture Therapy (BNCT)

Based on the previously reported potent and selective sulfone hydroxamate inhibitors SC-76276, SC-78080 (SD-2590), and SC-77964, potent MMP inhibitors have been designed and synthesized to append a boron-rich carborane cluster by employing click chemistry to target tumor cells that are known to upregulate gelatinases. Docking against MMP-2 suggests binding involving the hydroxamate zinc-binding group, key H-bonds by the sulfone moiety with the peptide backbone residues Leu82 and Leu83, and a hydrophobic interaction with the deep P1’ pocket. The more potent of the two triazole regioisomers exhibits an IC₅₀ of 3.7 nM versus MMP-2 and IC₅₀ of 46 nM versus MMP-9.     

Benzene polyphosphates as tools for cell signalling: inhibition of inositol 1,4,5-trisphosphate 5-phosphatase and interaction with the PH domain of protein kinase Balpha

Novel benzene polyphosphates were synthesised as inositol polyphosphate mimics and evaluated against type-I inositol 1,4,5-trisphosphate 5-phosphatase, which only binds soluble inositol polyphosphates, and against the PH domain of protein kinase Balpha (PKBalpha), which can bind both soluble inositol polyphosphates and inositol phospholipids. The most potent trisphosphate 5-phosphatase inhibitor is benzene 1,2,4-trisphosphate (2, IC(50) of 14 microM), a potential mimic of D-myo-inositol 1,4,5-trisphosphate, whereas the most potent tetrakisphosphate Ins(1,4,5)P(3) 5-phosphatase inhibitor is benzene 1,2,4,5-tetrakisphosphate, with an IC(50) of 4 microM. Biphenyl 2,3',4,5',6-pentakisphosphate (4) was the most potent inhibitor evaluated against type I Ins(1,4,5)P(3) 5-phosphatase (IC(50) of 1 microM). All new benzene polyphosphates are resistant to dephosphorylation by type I Ins(1,4,5)P(3) 5-phosphatase. Unexpectedly, all benzene polyphosphates studied bind to the PH domain of PKBalpha with apparent higher affinity than to type I Ins(1,4,5)P(3) 5-phosphatase. The most potent ligand for the PKBalpha PH domain, measured by inhibition of biotinylated diC(8)-PtdIns(3,4)P(2) binding, is biphenyl 2,3',4,5',6-pentakisphosphate (4, K(i)=27 nm). The approximately 80-fold enhancement of binding relative to parent benzene trisphosphate is explained by the involvement of a cation-pi interaction. These new molecular tools will be of potential use in structural and cell signalling studies.     

Synthesis,In Vitro,and Computational Studies of PTP1B Phosphatase Inhibitors Based on Oxovanadium(IV) and Dioxovanadium(V) Complexes

One of the main goals of recent bioinorganic chemistry studies has been to design and synthesize novel substances to treat human diseases. The promising compounds are metal-based and metal ion binding components such as vanadium-based compounds. The potential anticancer action of vanadium-based compounds is one of area of investigation in this field. In this study, we present five oxovanadium(IV) and dioxovanadium(V) complexes as potential PTP1B inhibitors with anticancer activity against the MCF-7 breast cancer cell line, the triple negative MDA-MB-231 breast cancer cell line, and the human keratinocyte HaCaT cell line. We observed that all tested compounds were effective inhibitors of PTP1B, which correlates with anticancer activity. [VO(dipic)(dmbipy)]·2 H₂O (Compound 4) and [VOO(dipic)](2-phepyH)·H₂O (Compound 5) possessed the greatest inhibitory effect, with IC₅₀ 185.4 ± 9.8 and 167.2 ± 8.0 nM, respectively. To obtain a better understanding of the relationship between the structure of the examined compounds and their activity, we performed a computer simulation of their binding inside the active site of PTP1B. We observed a stronger binding of complexes containing dipicolinic acid with PTP1B. Based on our simulations, we suggested that the studied complexes exert their activity by stabilizing the WPD-loop in an open position and limiting access to the P-loop.     

Screening of protease inhibitors as antiplasmodial agents. Part I: Aziridines and epoxides

A broad protease-based and cell-based screening of protease inhibitors yielded the aziridine-2-carboxylic acid derivative 2 a and the N-acylated aziridine-2,3-dicarboxylic acid derivatives 32 a and 34 b as the most potent inhibitors of falcipain-2 and falcipain-3 (IC(50) falcipain-2: 0.079-5.4 microM, falcipain-3: 0.25-39.8 microM). As the compounds also display in vitro activity against the P. falciparum parasite in the submicromolar and low micromolar range, these compound classes are leads for new antiplasmodial falcipain inhibitors.     

Eilatin Ru(II) complexes display anti-HIV activity and enantiomeric diversity in the binding of RNA

Eilatin-containing octahedral ruthenium complexes inhibit HIV-1 replication in CD4+ HeLa cells and in human peripheral blood monocytes with IC(50) values of approximately 1 microM. Similar metal complexes that lack eilatin display 15-100-fold lower anti-HIV activities. [Ru(bpy)(2)"pre-eilatin"](2+), a complex that contains a nonplanar analogue of eilatin, shows significantly lower nucleic acid binding and lower anti-HIV activity than eilatin complexes. This result indicates that the extended planar surface presented by eilatin is important for both activities. Rev peptide and ethidium bromide displacement assays are used to probe the nucleic acid affinity and specificity of Lambda- and Delta-[Ru(bpy)(2)eilatin](2+). Two HIV-1 RNA sites are compared and a significant binding preference for the Rev response element over the transactivation response region is found. Simple DNA duplexes show a consistent selectivity for Lambda-[Ru(bpy)(2)eilatin](2+) compared to Delta-[Ru(bpy)(2)eilatin](2+), while RNAs show more diverse enantiomeric selectivities.     

Discovery of potent inhibitors of PapG adhesins from uropathogenic Escherichia coli through synthesis and evaluation of galabiose derivatives

The synthesis of two galabioside (Galalpha1-4Gal) collections based on diversification at the O-1 and O-3' atoms is reported. The galabiosides were evaluated as inhibitors of hemagglutination of human erythrocytes by two strains of Escherichia coli that expressed the class I and class II PapG adhesins, respectively. The class I adhesin was found to prefer aromatic substituents both at the O-1 and the O-3' position of the galabiose disaccharide. One galabioside, p-methoxyphenyl [3-O-(m-nitrobenzyl)-alpha-D-galactopyranosyl]-(1-4)-beta-D-galactopyranoside], had an IC(50) value of 4.1 microM, which is the best inhibition of the class I adhesin to date. In the case of the class II adhesin, one inhibitor, 2-[(S)-2-methoxycarbonyl-2-acetamido-ethylthio]ethyl (3-O-3-[2-(methoxycarbonylphenylthio)propyl]-alpha-D-galactopyranosyl)-(1-4)-alpha-D-galactopyranoside, was found to have an IC(50) value of 68 microM, which is the best artificial inhibition of the class II adhesin reported so far with an affinity for the adhesin comparable to that of the natural tetrasaccharide ligand globotetraose.     

Synthesis and evaluation of 3-phenylpyrazolo[3,4-d]pyrimidine-peptide conjugates as Src kinase inhibitors

3-Phenylpyrazolo[3,4-d]pyrimidine (PhPP) derivatives substituted with an alkyl or aryl carboxylic acid at the N1-endocyclic amine, such as PhPP-CH(2)COOH (IC(50)=250 microM), and peptides Ac-CIYKYY (IC(50)=400 microM) and Ac-YIYGSFK (IC(50)=570 microM) were weak inhibitors of polyE(4)Y phosphorylation by active c-Src. A series of PhPP-peptide conjugates were synthesized using PhPP as an ATP mimic and CIYKYY or YIYGSFK as a peptide substrate to improve the inhibitory potency against active c-Src kinase. PhPP derivatives were attached to the N terminus or the side chain of amino acids in the peptide template. Two N-terminal substituted conjugates, PhPP-CH(2)CO-CIYKYY (IC(50)=0.38 microM) and PhPP-CH(2)CO-YIYGSFK (IC(50)=2.7 microM), inhibited the polyE(4)Y phosphorylation by active c-Src significantly higher than that of the parent compounds. The conjugation of PhPP with the peptides produced a synergistic inhibition effect possibly through creation of favorable interactions between the conjugate and the kinase domain as shown by molecular modeling studies.