6‑(1-Benzyl‑1<i>H</i>‑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 <b>6</b>, 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, <b>7a</b>–<b>y</b> and <b>8a</b>–<b>y</b>, 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 <b>7b</b>, <b>7u</b>, and <b>8g</b> were the most active against the RNase H activity of reverse-transcriptase, with IC₅₀ values of 3, 3, and 2.5 μM, respectively. Compound <b>8g</b> was also the most potent integrase inhibitor with an IC₅₀ value of 26 nM

Cell cycle.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell cycle analysis was performed after permeabilization using 7-AAD. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Nonspecific activity.

<p>HEK293 cells stably expressing Pdcd4_(mut39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b>. Luciferase activity is given relative to TPA-treated controls. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Proliferation.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 5 days with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (1 μM (green), 3 μM (violet), 10 μM (yellow), and 30 μM (red)). Confluency was assessed every 4 h in an IncuCyte instrument. Proliferation kinetics shown are representative for at least 3 independent experiments and represent means of 3 wells treated in parallel (* p<0.05, ** p<0.01, *** p<0.01).</p

Conformational analysis of compounds 1, 2 and 4.

<p>(A) A systematic approach was chosen to explore the conformational space around the three rotable bonds. (B-D) Global minimum energy conformations (<i>left panel</i>) and energy plots (<i>right panel</i>) of compounds <b>1</b> (B), <b>2</b> (C), and <b>4</b> (D).</p

Pdcd4 stabilizing activity.

<p>HEK293 cells stably expressing the Pdcd4 stability reporter Pdcd4_(39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b>. Pdcd4 stabilizing activity was determined relative to Δ(RLU_DMSO–RLU_TPA). Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Chemical structures.

<p>Structures of compounds <b>1</b> (1,2-bis(4-chlorophenyl)disulfide), <b>2</b> (4-chloro-<i>N</i>-(4-chlorophenyl)benzamide), <b>3</b> ((<i>E</i>)-1,2-bis(4-chlorophenyl)diazene), <b>4</b> (1,2-bis(4-chlorophenyl)hydrazine), <b>5</b> (1,2-bis(4-methoxyphenyl)disulfide), and <b>6</b> (1,2-bis(4-nitrophenyl)disulfide).</p

Viability.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell death analysis was performed using Annexin V and 7-AAD co-staining. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Design, Synthesis, and Structure–Activity Relationship of <i>N</i>‑Arylnaphthylamine Derivatives as Amyloid Aggregation Inhibitors

Dyes like CR are able to inhibit the aggregation of Aβ fibrils. Thus, a screening of a series of dyes including ABBB (<b>1</b>) was performed. Its main component <b>2</b> tested in an in vitro assay (i.e., ThT assay) showed good potency at inhibiting fibrils association. Congeners <b>4</b>–<b>9</b> have been designed and synthesized as inhibitors of Aβ aggregation. A number of these newly synthesized compounds have been found to be active in the ThT assay with IC₅₀ of 1–57.4 μM. The most potent compound of this series, <b>4k</b>, showed micromolar activity in this test. Another potent derivative <b>4q</b> (IC₅₀ = 5.6 μM) rapidly crossed the blood–brain barrier, achieving whole brain concentrations higher than in plasma. So <b>4q</b> could be developed to find novel potent antiaggregating βA agents useful in Alzheimer disease as well as other neurological diseases characterized by deposits of amyloid aggregates

New Nucleotide-Competitive Non-Nucleoside Inhibitors of Terminal Deoxynucleotidyl Transferase: Discovery, Characterization, and Crystal Structure in Complex with the Target

Terminal deoxynucletidyl transferase (TdT) is overexpressed in some cancer types, where it might compete with pol μ during the mutagenic repair of double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. Here we report the discovery and characterization of pyrrolyl and indolyl diketo acids that specifically target TdT and behave as nucleotide-competitive inhibitors. These compounds show a selective toxicity toward MOLT-4 compared to HeLa cells that correlate well with in vitro selectivity for TdT. The binding site of two of these inhibitors was determined by cocrystallization with TdT, explaining why these compounds are competitive inhibitors of the deoxynucleotide triphosphate (dNTP). In addition, because of the observed dual localization of the phenyl substituent, these studies open the possibility of rationally designing more potent compounds