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Supplementary Figures 1-6 from Enigmol: A Novel Sphingolipid Analogue with Anticancer Activity against Cancer Cell Lines and In vivo Models for Intestinal and Prostate Cancer

Supplementary Methods and Results from Enigmol: A Novel Sphingolipid Analogue with Anticancer Activity against Cancer Cell Lines and In vivo Models for Intestinal and Prostate Cancer

Supplementary Material from Enigmol: A Novel Sphingolipid Analogue with Anticancer Activity against Cancer Cell Lines and In vivo Models for Intestinal and Prostate Cancer

The orally bioavailable 1-deoxy-sphingosine analog, Enigmol, has demonstrated anticancer activity in numerous in vivo settings. However, as no Enigmol analog with enhanced potency in vitro has been identified, a new strategy to improve efficacy in vivo by increasing tumor uptake was adopted. Herein, synthesis and biological evaluation of two novel fluorinated Enigmol analogs, CF3-Enigmol and CF2-Enigmol, are reported. Each analog was equipotent to Enigmol in vitro, but achieved higher plasma and tissue levels than Enigmol in vivo. Although plasma and tissue exposures were anticipated to trend with fluorine content, CF2-Enigmol absorbed into tissue at strikingly higher concentrations than CF3-Enigmol. Using mouse xenograft models of prostate cancer, we also show that CF3-Enigmol underperformed Enigmol-mediated inhibition of tumor growth and elicited systemic toxicity. By contrast, CF2-Enigmol was not systemically toxic and demonstrated significantly enhanced antitumor activity as compared to Enigmol.

Group A streptococcus (GAS) is a Gram-positive bacterium, which can cause multiple types of disease from mild infections of skin and throat to invasive and life-threatening infections. Recently RNase J1 and J2 were found to be essential for the growth of GAS. In order to identify inhibitors against RNase J1/J2, homology models of both the ligand-free apo-form and the ligand-bound holo-form complexes were constructed as templates for high-throughput virtual screening (HTVS). A focused small molecule library and the commercially available Maybridge database were employed as sources for potential inhibitors. A cell-based biological assay identified two compounds with 10 μM MIC activity.

Despite highly active antiretroviral therapy (HAART), human immunodeficiency virus-1 (HIV-1) continues to elude eradication. Even in the setting of plasma virologic suppression, HIV-1 RNA can be found in peripheral blood mononuclear cells (PBMCs), genital secretions, gastrointestinal lymphoid tissue, and cerebrospinal fluid (CSF).1,2 Limited penetration of antiretrovirals (ARVs) into these sites may play a role in viral persistence.2,3 HIV-1 invades the central nervous system (CNS) early after infection via macrophages, monocytes, and dendritic cells.4 Over time, HIV-associated neurocognitive disorders (HAND) can develop,5 contributing significantly to morbidity6 and early mortality.7 While severity of HAND has decreased, prevalence remains unchanged since the pre-HAART era: 36.2–44.8%,6 prompting a concern for suboptimal CNS control of HIV. This is supported by reports of elevated neopterin, an inflammatory biomarker, and measurable HIV RNA in the CSF of patients on long term HAART,8,9 both predictive markers for development of HIV-associated dementia.10,11 Some studies suggest that limited CNS penetration by ARVs is associated with HAND.12,13 Molecular size, lipophilicity, affinity for efflux pump transporters, and degree of plasma protein binding are factors that impact drug CNS penetration.14 Lipophilic drugs, including HIV protease inhibitors (PIs), are highly bound to plasma protein, predominantly alpha1-acid glycoprotein (AAG). The plasma unbound (free) drug component is considered pharmacologically active and readily crosses the blood–brain and blood–CSF barriers15,16 therefore highly bound drugs such as PIs may have limited CNS penetration. Two PIs, atazanavir (ATV) and darunavir (DRV), are recommended by the Department of Health and Human Services (DHHS) as part of first-line once daily regimens for ARV-naive patients when boosted with ritonavir (RTV) and given with a backbone of tenofovir disoproxil fumarate/emtricitabine (TDF/FTC).17 Both PIs have characteristics that could impede tissue and CNS penetration, including large molecular size and affinity for efflux pump transporters. However, they differ in their affinity for plasma AAG: ATV is 86% plasma protein bound in vitro whereas DRV is 95% bound.18,19 The degree to which they successfully penetrate the CNS remains unclear. Best et al showed low or undetectable CSF ATV levels in nearly 25% of study participants.20 but measured only total drug concentrations with a lower quantitation limit of 5 ng/mL. In contrast, 3 prior studies have shown that DRV can attain drug concentrations in the CSF exceeding IC50 21–23 ; however, the single study measuring unbound drug concentrations22 examined participants receiving twice daily DRV rather than the DHHS recommended once daily dosing schedule for patients without history of DRV resistance.17 Additionally, measuring random total drug concentrations rather than troughs limited all previous studies. To address these limitations, we evaluated the CSF penetration of once daily DRV relative to ATV, both boosted with RTV and administered with the same background TDF/FTC regimen. We hypothesized that ATV, a PI with lower degree of plasma protein binding, would achieve a higher CSF/plasma unbound drug concentration ratio than DRV. In addition, we compared unbound CSF PI concentrations to their drug-specific IC50 and assessed relationships with CSF HIV-1 RNA and neopterin.

Fumonisin B(1) (FB(1)) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB(1) increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C(18)H(40)NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH(3)CH=NH (+)(2)) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-(13)C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK(1) cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-(13)C]palmitate into 1-[(13)C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[(13)C]deoxySa. 1-DeoxySa was elevated in FB(1)-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK(1) and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB(1), substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation.

“Sphingosin” was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of “lipides derived from sphingosine as sphingolipides.” This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the α,ω-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds.

HIV-1 protease inhibitors (PIs) exhibit different protein binding affinities and achieve variable plasma and tissue concentrations. Degree of plasma protein binding may impact central nervous system penetration. This cross-sectional study assessed cerebrospinal fluid (CSF) unbound PI concentrations, HIV-1 RNA, and neopterin levels in subjects receiving either ritonavir-boosted darunavir (DRV), 95% plasma protein bound, or atazanavir (ATV), 86% bound. Unbound PI trough concentrations were measured using rapid equilibrium dialysis and liquid chromatography/tandem mass spectrometry. Plasma and CSF HIV-1 RNA and neopterin were measured by Ampliprep/COBAS® Taqman® 2.0 assay (Roche) and enzyme-linked immunosorbent assay (ALPCO), respectively. CSF/plasma unbound drug concentration ratio was higher for ATV, 0.09 [95% confidence interval (CI) 0.06–0.12] than DRV, 0.04 (95%CI 0.03–0.06). Unbound CSF concentrations were lower than protein adjusted wild-type inhibitory concentration-50 (IC50) in all ATV and 1 DRV-treated subjects (P < 0.001). CSF HIV-1 RNA was detected in 2/15 ATV and 4/15 DRV subjects (P = 0.65). CSF neopterin levels were low and similar between arms. ATV relative to DRV had higher CSF/plasma unbound drug ratio. Low CSF HIV-1 RNA and neopterin suggest that both regimens resulted in CSF virologic suppression and controlled inflammation.

INTRODUCTION AND OBJECTIVES: Retroviral replicating vectors (RRVs) represent a novel gene delivery platform that can achieve efficient transduction of solid tumors through virus propagation within actively dividing cancer cells. FDA has already approved a first-in-human clinical trial of RRV-mediated prodrug-activator gene therapy, which is currently on-going. To further improve their tumor specificity and safety profile in prostate cancer, we have developed RRVs regulated by prostate-specific promoters. METHODS: Prostate-specific promoter sequences ARR2PB (synthetic variant of androgen-dependent probasin promoter) or PSES (androgen-independent, synthetic PSA/PSMA composite promoter) were used to replace the wild-type virus promoter in RRV expressing either the GFP reporter gene (RRVs ACE-GFP-ARR2PB and ACEGFP-PSES, respectively) or the yeast cytosine deaminase prodrugactivator gene, which converts the prodrug 5-fluorocytosine into the active drug 5-fluorouracil (RRVs ACE-CD-ARR2PB and ACE-CDPSES, respectively). RRV replication kinetics and selectivity were examined in prostate (LNCaP, MDA-PCa-2b and PC3) and non-prostate (293T) cell lines and primary human T cells, and in LNCaP subcutaneous xenograft tumors. Cytotoxic effects of prodrug-activator gene therapy were also examined both in vitro and in vivo. Systemic biodistribution was examined by qPCR in male athymic mice after intravenous RRV injection. RESULTS: RRV ACE-GFP-ARR2PB showed efficient replication only in androgen receptor-positive prostate cancer cells, and only in the presence of androgen. RRV ACE-GFP-PSES replication was highly specific for PSA/PSMA-positive prostate cancer cells, with or without androgen. In primary T cells, transgene expression and replication of both prostate-targeted RRVs were highly suppressed compared to non-targeted wild type RRV. In vivo, prostate-targeted RRVs showed efficient spread within LNCaP tumors, and both ACE-CDARR2PB and ACE-CD-PSES achieved effective tumor growth inhibition even after initial transduction at very low levels. Systemic biodistribution of prostate-targeted RRVs was highly restricted to tumor tissues. CONCLUSIONS: ARR2PBand PSES-targeted RRVs exhibit selective replication and efficient gene delivery in prostate cancer models, achieving potent therapeutic efficacy in vitro and in vivo. In particular, PSES-targeted RRVs may be highly useful for gene therapy in castration-resistant prostate cancer. The ability to target RRVs specifically to cancer cells will limit and control the replicative process, and minimize risk to normal cells.

Sphingoid bases are cytotoxic for many cancer cell lines and are thought to contribute to suppression of intestinal tumorigenesis in vivo by ingested sphingolipids. This study explored the behavior of a sphingoid base analogue, (2S,3S,5S)-2-amino-3,5-dihydroxyoctadecane (Enigmol), that cannot be phosphorylated by sphingosine kinases and is slowly N-acylated and therefore is more persistent than natural sphingoid bases. Enigmol had potential anticancer activity in a National Cancer Institute (NCI-60) cell line screen and was confirmed to be more cytotoxic and persistent than naturally occurring sphingoid bases using HT29 cells, a colon cancer cell line. Although the molecular targets of sphingoid bases are not well delineated, Enigmol shared one of the mechanisms that has been found for naturally occurring sphingoid bases: normalization of the aberrant accumulation of β-catenin in the nucleus and cytoplasm of colon cancer cells due to defect(s) in the adenomatous polyposis coli (APC)/β-catenin regulatory system. Enigmol also had antitumor efficacy when administered orally to Min mice, a mouse model with a truncated APC gene product (C57Bl/6JMin/+ mice), decreasing the number of intestinal tumors by half at 0.025% of the diet (w/w), with no evidence of host toxicity until higher dosages. Enigmol was also tested against the prostate cancer cell lines DU145 and PC-3 in nude mouse xenografts and suppressed tumor growth in both. Thus, Enigmol represents a novel category of sphingoid base analogue that is orally bioavailable and has the potential to be effective against multiple types of cancer. Mol Cancer Ther; 10(4); 648–57. ©2011 AACR.

"Sphingosin" was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of "lipides derived from sphingosine as sphingolipides." This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the alpha,omega-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds.

For a wide variety of microbial pathogens, the outcome of the infection is indeterminate. In some individuals the microbe is cleared, but in others it establishes a chronic infection, and the factors that tip this balance are often unknown. In a widely used model of chronic viral infection, C57BL/6 mice clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), but the clone 13 strain persists. Here we show that the Armstrong strain induces a profound lymphopenia at days 1-3 after infection, but the clone 13 strain does not. If we transiently augment lymphopenia by treating the clone-13-infected mice with the drug FTY720 at days 0-2 after infection, the mice successfully clear the infection by day 30. Clearance does not occur when CD4 T cells are absent at the time of treatment, indicating that the drug is not exerting direct antiviral effects. Notably, FTY720 treatment of an already established persistent infection also leads to viral clearance. In both models, FTY720 treatment preserves or augments LCMV-specific CD4 and CD8 T-cell responses, a result that is counter-intuitive because FTY720 is generally regarded as a new immunosuppressive agent. Because FTY720 targets host pathways that are completely evolutionarily conserved, our results may be translatable into new immunotherapies for the treatment of chronic microbial infections in humans.

Safingol [(2S,3S)-2-amino-1,3-octadecanediol] is an unnatural l-threo-stereoisomer of sphinganine that is cytotoxic for cancer cells in culture and is being tested in phase 1 human clinical trials. To determine if safingol can be absorbed orally and if it affects prostate cancer in a mouse strain used in prostate cancer studies, safingol was fed to TRAMP (transgenic adenocarcinoma of mouse prostate) mice for 2 weeks at 0.0125% to 0.1% w/w of the diet. Analysis of safingol and safingol metabolites in blood and tissues by liquid chromatography electrospray ionization tandem mass spectrometry revealed uptake in tissue and extensive conversion of safingol to N-acyl species (comparable to natural "ceramides") and mono-, di-, and tri-N-methyl metabolites that have not been observed previously. Safingol caused significant hepatotoxicity at all dosages, as reflected in elevated liver alanine aminotransferase, and at the highest dose (0.1 %) caused changes in liver histology (appearance of autophagosomal vacuoles) and renal toxicity (based on elevation of blood urea nitrogen) and decreases in packed blood cell volume and body weight. Safingol did not inhibit the prostate pre-neoplastic lesion (prostate intraepithelial neoplasia) in TRAMP mice; however, additional studies at lower dosages for longer time were not pursued due to host toxicity. Safingol and its N-methyl metabolites were cytotoxic to both a human prostate cell line (DU145) and mouse BALB 3T3 cells; therefore, the host and potential antitumor toxicity may be due to multiple molecular species of safingol.

The authors wish to retract this Letter on the grounds that they have been unable to repeat key observations of the paper. Using currently available stocks of virus (LCMV clone 13) and drug (FTY720), they no longer observe that transient drug treatment of infected mice leads to immune-mediated clearance of virus.