Total Syntheses of Phorbol and 11 Tigliane Diterpenoids and Their Evaluation as HIV Latency-Reversing Agents.

Tigliane diterpenoids possess exceptionally complex structures comprising common 5/7/6/3-membered ABCD-rings and disparate oxygen functionalities. While tiglianes display a wide range of biological activities, compounds with HIV latency-reversing activity can eliminate viral reservoirs, thereby serving as promising leads for new anti-HIV agents. Herein, we report collective total syntheses of phorbol (13) and 11 tiglianes 14-24 with various acylation patterns and oxidation states, and their evaluation as HIV latency-reversing agents. The syntheses were strategically divided into five stages to increase the structural complexity. First, our previously established sequence enabled the expeditious preparation of ABC-tricycle 9 in 15 steps. Second, hydroxylation of 9 and ring-contractive D-ring formation furnished phorbol (13). Third, site-selective attachment of two acyl groups to 13 produced four phorbol diesters 14-17. Fourth, the oxygen functionalities were regio- and stereoselectively installed to yield five tiglianes 18-22. Fifth, further oxidation to the most densely oxygenated acerifolin A (23) and tigilanol tiglate (24) was realized through organizing a 3D shape of the B-ring. Assessment of the HIV latency-reversing activities of the 12 tiglianes revealed seven tiglianes (14-17 and 22-24) with 20- to 300-fold improved efficacy compared with prostratin (12), a representative latency-reversing agent. Therefore, the robust synthetic routes to a variety of tiglianes with promising activities devised in this study provide opportunities for advancing HIV eradication strategies.

Synthesis and Biological Evaluation of Simplified Ansellone Analogues with Lipophilic Side Chains as HIV Latency-Reversing Agents.

Structurally simplified analogues of ansellone A, in which the decalin skeleton is replaced with a lipophilic chain, were prepared and their HIV latency-reversing activities biologically evaluated. In particular, two analogues bearing ether and alkenyl side chains, respectively, showed comparable activities to that of ansellone A. Each of the simplified compounds was easily synthesized using Prins cyclisation chemistry.

Total Synthesis of Ansellone G and Phorbadione.

The first total synthesis of marine sesterterpenoid ansellone G (2) was accomplished. This strategy utilizes the Prins cyclization reaction of a chloro-substituted homoallyl alcohol to synthesize the hydrobenzopyran skeleton. The preintroduction of the chloro groups facilitated the functional group transformation for 2 after constructing the carbon framework. Furthermore, we also successfully synthesized phorbadione (3) by dehydrating the tertiary alcohol. The HIV latency-reversing activity of the synthesized 2, 3, and deacetylated 2 was also evaluated.

Glucose-dependent aerobic glycolysis contributes to recruiting viral components into HIV-1 particles to maintain infectivity.

The cellular environment affects optimal viral replication because viruses cannot replicate without their host cells. In particular, metabolic resources such as carbohydrates, lipids, and ATP are crucial for viral replication, which is sensitive to cellular metabolism. Intriguingly, recent studies have demonstrated that human immunodeficiency virus type 1 (HIV-1) infection induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis in CD4+ T cells to produce the virus efficiently. However, the importance of aerobic glycolysis in maintaining the quality of viral components and viral infectivity has not yet been fully investigated. Here, we show that aerobic glycolysis is necessary not only to override the inhibitory effect of virion-incorporated glycolytic enzymes, but also to maintain the enzymatic activity of reverse transcriptase and the adequate packaging of envelope proteins into HIV-1 particles. To investigate the effect of metabolic remodeling on the phenotypic properties of HIV-1 produced by infected cells, we replaced glucose with galactose in the culture medium because the cells grown in galactose-containing medium are forced to carry out oxidative metabolism instead of aerobic glycolysis. We found that the packaging levels of glyceraldehyde 3-phosphate dehydrogenase, alpha-enolase and pyruvate kinase muscle type 2, which decrease HIV-1 infectivity by packaging into viral particles, are increased in progeny viruses produced by the cells grown in galactose-containing medium. Furthermore, we found that the entry and reverse transcription efficiency of the progeny viruses were reduced, which was caused by a decrease in the enzymatic activity of reverse transcriptase in the viral particles and a decrease in the packaging levels of envelope proteins and reverse transcriptase. These results indicate that the aerobic glycolysis environment in HIV-1-infected cells may contribute to the quality control of viruses.

Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration.

BACKGROUND: A protein exhibiting more than one biochemical function is termed a moonlighting protein. Glycolytic enzymes are typical moonlighting proteins, and these enzymes control the infection of various viruses. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and alpha-enolase (ENO1) are incorporated into human immunodeficiency virus type 1 (HIV-1) particles from viral producer cells and suppress viral reverse transcription independently each other. However, it remains unclear whether these proteins expressed in viral target cells affect the early phase of HIV-1 replication. RESULTS: Here we show that the GAPDH expression level in viral target cells does not affect the early phase of HIV-1 replication, but ENO1 has a capacity to suppress viral integration in viral target cells. In contrast to GAPDH, suppression of ENO1 expression by RNA interference in the target cells increased viral infectivity, but had no effect on the expression levels of the HIV-1 receptors CD4, CCR5 and CXCR4 and on the level of HIV-1 entry. Quantitative analysis of HIV-1 reverse transcription products showed that the number of copies of the late products (R/gag) and two-long-terminal-repeat circular forms of viral cDNAs did not change but that of the integrated (Alu-gag) form increased. In contrast, overexpression of ENO1 in viral target cells decreased viral infectivity owing to the low viral integration efficiency. Results of subcellular fractionation experiments suggest that the HIV integration at the nucleus was negatively regulated by ENO1 localized in the nucleus. In addition, the overexpression of ENO1 in both viral producer cells and target cells most markedly suppressed the viral replication. CONCLUSIONS: These results indicate that ENO1 in the viral target cells prevents HIV-1 integration. Importantly, ENO1, but not GAPDH, has the bifunctional inhibitory activity against HIV-1 replication. The results provide and new insights into the function of ENO1 as a moonlighting protein in HIV-1 infection.

Total Synthesis and Biological Evaluation of the Potent HIV Latency-Reversing Agent Ansellone A and its Analogues.

The total synthesis and biological evaluation of the marine sesterterpenoid ansellone A (1), an HIV latency-reversing agent, and its analogues are reported. The key to the success of this synthetic route is a Prins cyclization reaction enabled by the strategic use of the TfO group for stabilization of the acid-labile tertiary allylic alcohol. The SAR study found the alcohol analogue to exhibit more potent activity than 1.