Diaryl Disulfides as Novel Stabilizers of Tumor Suppressor Pdcd4.

The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70S6K1 dependent phosphorylation of Pdcd4 followed by ß-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2-4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted.

Discovery of N-aryl-naphthylamines as in vitro inhibitors of the interaction between HIV integrase and the cofactor LEDGF/p75.

A series of N-aryl-naphthylamines, exemplified by the structures 11-16, were chosen for an in-house library screening to assay their ability to disrupt the interaction between the LEDGF cofactor and the HIV integrase. Structure modification led also to design and synthesize new compounds 17a-f. Compounds 11e,h,k,n, 13b, and 14 showed good activity in AlphaScreen assay. The most active compound 11e (IC50 = 2.5 µM) was selected for molecular modeling studies and showed a binding mode similar to the one of the known LEDGIN 8.

N-Substituted Quinolinonyl Diketo Acid Derivatives as HIV Integrase Strand Transfer Inhibitors and Their Activity against RNase H Function of Reverse Transcriptase.

Bifunctional quinolinonyl DKA derivatives were first described as nonselective inhibitors of 3'-processing (3'-P) and strand transfer (ST) functions of HIV-1 integrase (IN), while 7-aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also displayed activity against ribonuclease H (RNase H). In this study, we describe the design, synthesis, and biological evaluation of new quinolinonyl diketo acid (DKA) derivatives characterized by variously substituted alkylating groups on the nitrogen atom of the quinolinone ring. Removal of the second DKA branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a fine-tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity for IN ST activity. In vitro, the most potent compound was 11j (IC50 = 10 nM), while the most active compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against RNase H was negligible, with only a few compounds active at concentrations higher than 10 µM. The binding mode of the most potent IN inhibitor 11j within the IN catalytic core domain (CCD) is described as well as its binding mode within the RNase H catalytic site to rationalize its selectivity.

Structure-activity relationship of pyrrolyl diketo acid derivatives as dual inhibitors of HIV-1 integrase and reverse transcriptase ribonuclease H domain.

The development of HIV-1 dual inhibitors is a highly innovative approach aimed at reducing drug toxic side effects as well as therapeutic costs. HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) are both selective targets for HIV-1 chemotherapy, and the identification of dual IN/RNase H inhibitors is an attractive strategy for new drug development. We newly synthesized pyrrolyl derivatives that exhibited good potency against IN and a moderate inhibition of the RNase H function of RT, confirming the possibility of developing dual HIV-1 IN/RNase H inhibitors and obtaining new information for the further development of more effective dual HIV-1 inhibitors.

Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives.

HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.

Basic quinolinonyl diketo acid derivatives as inhibitors of HIV integrase and their activity against RNase H function of reverse transcriptase.

A series of antiviral basic quinolinonyl diketo acid derivatives were developed as inhibitors of HIV-1 IN. Compounds 12d,f,i inhibited HIV-1 IN with IC50 values below 100 nM for strand transfer and showed a 2 order of magnitude selectivity over 3'-processing. These strand transfer selective inhibitors also inhibited HIV-1 RNase H with low micromolar potencies. Molecular modeling studies based on both the HIV-1 IN and RNase H catalytic core domains provided new structural insights for the future development of these compounds as dual HIV-1 IN and RNase H inhibitors.

6-(1-Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H, synthesized by a parallel synthesis approach.

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 µM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.

Discovery and pharmacological profile of new 1H-indazole-3-carboxamide and 2H-pyrrolo[3,4-c]quinoline derivatives as selective serotonin 4 receptor ligands.

Since the discovery of the serotonin 4 receptor (5-HT(4)R), a large number of receptor ligands have been studied. The safety concerns and the lack of market success of these ligands have mainly been attributed to their lack of selectivity. In this study we describe the discovery of N-[(4-piperidinyl)methyl]-1H-indazole-3-carboxamide and 4-[(4-piperidinyl)methoxy]-2H-pyrrolo[3,4-c]quinoline derivatives as new 5-HT(4)R ligands endowed with high selectivity over the serotonin 2A receptor and human ether-a-go-go-related gene potassium ion channel. Within these series, two molecules (11 ab and 12 g) were identified as potent and selective 5-HT(4)R antagonists with good in vitro pharmacokinetic properties. These compounds were evaluated for their antinociceptive action in two analgesia animal models. 12 g showed a significant antinociceptive effect in both models and is proposed as an interesting lead compound as a 5-HT(4)R antagonist with analgesic action.

Design, synthesis, and structure-activity relationship of N-arylnaphthylamine derivatives as amyloid aggregation inhibitors.

Dyes like CR are able to inhibit the aggregation of Aß fibrils. Thus, a screening of a series of dyes including ABBB (1) was performed. Its main component 2 tested in an in vitro assay (i.e., ThT assay) showed good potency at inhibiting fibrils association. Congeners 4-9 have been designed and synthesized as inhibitors of Aß aggregation. A number of these newly synthesized compounds have been found to be active in the ThT assay with IC(50) of 1-57.4 µM. The most potent compound of this series, 4k, showed micromolar activity in this test. Another potent derivative 4q (IC(50) = 5.6 µM) rapidly crossed the blood-brain barrier, achieving whole brain concentrations higher than in plasma. So 4q could be developed to find novel potent antiaggregating ßA agents useful in Alzheimer disease as well as other neurological diseases characterized by deposits of amyloid aggregates.

Effects of polyphenol compounds on influenza A virus replication and definition of their mechanism of action.

A set of polyphenol compounds was synthesized and assayed for their ability in inhibiting influenza A virus replication. A sub-set of them showed low toxicity. The best compounds within this sub-set were 4 and 6g, which inhibited the viral replication in a dose-dependent manner. The antiviral activity of these molecules was demonstrated to be caused by their interference with intracellular pathways exploited for viral replication: (1) MAP kinases controlling nuclear-cytoplasmic traffic of viral ribonucleoprotein complex; (2) redox-sensitive pathways, involved in maturation of viral hemagglutinin protein.