Total Synthesis of Scytonemide A Employing Weinreb AM Solid-Phase Resin

The human 20S proteasome inhibitor scytonemide A (<b>1</b>), a macrocyclic imine originally isolated from the cyanobacterium <i>Scytonema hofmanni</i>, was synthesized via a biomimetic solid-phase peptide synthesis (SPPS) approach employing the Weinreb AM resin. Utilizing this approach, cyclization of the protected heptapeptide via formation of the imine bond occurred spontaneously upon cleavage from the resin in the presence of a reducing agent and subsequent aqueous workup. The final deprotection step necessary to produce the natural product was accomplished under slightly basic conditions, facilitating cleavage of the silyl ether group while leaving the macrocycle intact. Purification of the synthetic scytonemide A was accomplished via normal-phase flash column chromatography, potentially facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the ¹H NMR spectrum

The Competitive Interplay between Allosteric HIV‑1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV‑1 Replication Adversely Affects Inhibitor Potency

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC₅₀ for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein–protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner <i>in vitro</i>. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC₅₀ values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication

A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

<div><p>The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies <i>in vitro</i>. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC₅₀ of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.</p></div

Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to HIV-1 IN CCD.

<p>The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.</p

Chemical structures of ALLINIs and MINIs.

<p>Chemical structures of ALLINIs and MINIs.</p

Activities of HIV-1 IN inhibitors.

<p>Data for IC₅₀ and EC₅₀ are given as the mean ± SD from at least three independent experiments.</p><p>CC₅₀ values of >100 µM indicates that the respective inhibitors were not cytotoxic at the tested concentrations upto 100 µM.</p>a<p>BI-1001 EC₅₀ value and the assay method have been described elsewhere <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004171#ppat.1004171-Kessl3" target="_blank">[33]</a>.</p

Virions produced in the presence of KF116 are defective in reverse transcription.

<p>(<b>A–E</b>) VSV-G pseudotyped HIV-1-Luc produced in the presence of DMSO, 1 µM KF116, or 1 µM RAL were used to infect HEK293T cells. Infected cells were harvested at the indicated times and subjected to quantitative PCR (qPCR) or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for (<b>A</b>) early reverse transcription (Early RT), (<b>B</b>) late reverse transcription (Late RT) and (<b>C</b>) 2-LTR circles (2-LTRs) products. (<b>D</b>) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) control at 7 days post-infection. (<b>E</b>) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD (<i>n</i> = 3). (<b>F–J</b>) HEK293T cells were treated with DMSO, 1 µM KF116, or 1 µM RAL and then infected with VSV-G pseudotyped HIV-1-Luc. Infected cells were harvested at the indicated times and subjected to qPCR or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for (<b>F</b>) early reverse transcription (Early RT), (<b>G</b>) late reverse transcription (Late RT) and (<b>H</b>) 2-LTR circles (2-LTRs) products. (<b>I</b>) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) sample at 7 days post-infection. (<b>J</b>) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD (<i>n</i> = 3).</p

LEDGF/p75 expression does not affect KF116 potency during late stage of HIV-1 replication.

<p>(<b>A</b>) Equivalent whole cell lysates from the clonal TALEN-derived <i>PSIP</i>1 KO cell line (indicated as “KO”) and parental wild type HEK293T cell line (indicated as “WT”) were subjected to SDS-PAGE and immunoblotted for LEDGF/p75 and a GAPDH control to verify knockdown of LEDGF/p75 protein. (<b>B</b>) Dose-response curves representing the antiviral assays performed in WT or KO cell lines under the indicated conditions of drug treatment. For producer cell treatment, the VSV-G pseudotyped HIV-1-Luc progeny virions were prepared in the indicated cell line in the presence of KF116 and were then used to infect untreated HEK293T cells. For target cell treatment, KF116 was added directly to the indicated cell line and the cells were infected with untreated VSV-G pseudotyped HIV-1-Luc virions. (<b>C</b>) EC₅₀ values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.</p

Bioactive Flavaglines and Other Constituents Isolated from <i>Aglaia perviridis</i>

Eight new compounds, including two cyclopenta­[<i>b</i>]­benzopyran derivatives (<b>1</b>, <b>2</b>), two cyclopenta­[<i>b</i>]­benzofuran derivatives (<b>3</b>, <b>4</b>), three cycloartane triterpenoids (<b>5</b>–<b>7</b>), and an apocarotenoid (<b>8</b>), together with 16 known compounds, were isolated from the chloroform-soluble partitions of separate methanol extracts of a combination of the fruits, leaves, and twigs and of the roots of <i>Aglaia perviridis</i> collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29) and facilitated with an LC/MS dereplication procedure. The structures of the new compounds (<b>1</b>–<b>8</b>) were determined on the basis of spectroscopic data interpretation. The Mosher ester method was employed to determine the absolute configurations of <b>5</b>–<b>7</b>, and the absolute configuration of the 9,10-diol unit of compound <b>8</b> was established by a dimolybdenum tetraacetate [Mo₂(AcO)₄] induced circular dichroism procedure. Seven known rocaglate derivatives (<b>9</b>–<b>15</b>) exhibited significant cytotoxicity against the HT-29 cell line, with rocaglaol (<b>9</b>) being the most potent (ED₅₀ 0.0007 μM). The new compounds <b>2</b>–<b>4</b> were also active against this cell line, with ED₅₀ values ranging from 0.46 to 4.7 μM. The cytotoxic compounds were evaluated against a normal colon cell line, CCD-112CoN. In addition, the new compound perviridicin B (<b>2</b>), three known rocaglate derivatives (<b>9</b>,<b> 11</b>, <b>12</b>), and a known sesquiterpene, 2-oxaisodauc-5-en-12-al (<b>17</b>), showed significant NF-κB (p65) inhibitory activity in an ELISA assay

KF116 selectively impairs the late stage of HIV-1 replication.

<p>(<b>A</b>) Dose-response curves for KF116 antiviral activities during early stage, late stage or one full replication cycle. For early stage experiments, KF116 was added directly to the target cells and then these cells were infected with untreated virions. For late stage experiments, the progeny virions were prepared in the presence of KF116 and were then used to infect untreated target cells. For one full replication cycle experiments, KF116 was added to both producer and target cells. (<b>B</b>) EC₅₀ values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.</p