Lipid sulfoxide polymers as potential inhalable drug delivery platforms with differential albumin binding affinity.

Inhalable nanomedicines are increasingly being developed to optimise the pharmaceutical treatment of respiratory diseases. Large lipid-based nanosystems at the forefront of the inhalable nanomedicines development pipeline, though, have a number of limitations. The objective of this study was, therefore, to investigate the utility of novel small lipidated sulfoxide polymers based on poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA) as inhalable drug delivery platforms with tuneable membrane permeability imparted by differential albumin binding kinetics. Linear PMSEA (5 kDa) was used as a hydrophilic polymer backbone with excellent anti-fouling and stealth properties compared to poly(ethylene glycol). Terminal lipids comprising single (1C2, 1C12) or double (2C12) chain diglycerides were installed to provide differing affinities for albumin and, by extension, albumin trafficking pathways in the lungs. Albumin binding kinetics, cytotoxicity, lung mucus penetration and cellular uptake and permeability through key cellular barriers in the lungs were examined in vitro. The polymers showed good mucus penetration and no cytotoxicity over 24 h at up to 1 mg ml-1. While 1C2-showed no interaction with albumin, 1C12-PMSEA and 2C12-PMSEA bound albumin with KD values of approximately 76 and 10 µM, respectively. Despite binding to albumin, 2C12-PMSEA showed reduced cell uptake and membrane permeability compared to the smaller polymers and the presence of albumin had little effect on cell uptake and membrane permeability. While PMSEA strongly shielded these lipids from albumin, the data suggest that there is scope to tune the lipid component of these systems to control membrane permeability and cellular interactions in the lungs to tailor drug disposition in the lungs.

The Arylamine N-Acetyltransferases as Therapeutic Targets in Metabolic Diseases Associated with Mitochondrial Dysfunction.

In humans, there are two arylamine N-acetyltransferase genes that encode functional enzymes (NAT1 and NAT2) as well as one pseudogene, all of which are located together on chromosome 8. Although they were first identified by their role in the acetylation of drugs and other xenobiotics, recent studies have shown strong associations for both enzymes in a variety of diseases, including cancer, cardiovascular disease, and diabetes. There is growing evidence that this association may be causal. Consistently, NAT1 and NAT2 are shown to be required for healthy mitochondria. This review discusses the current literature on the role of both NAT1 and NAT2 in mitochondrial bioenergetics. It will attempt to relate our understanding of the evolution of the two genes with biologic function and then present evidence that several major metabolic diseases are influenced by NAT1 and NAT2. Finally, it will discuss current and future approaches to inhibit or enhance NAT1 and NAT2 activity/expression using small-molecule drugs. SIGNIFICANCE STATEMENT: The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 share common features in their associations with mitochondrial bioenergetics. This review discusses mitochondrial function as it relates to health and disease, and the importance of NAT in mitochondrial function and dysfunction. It also compares NAT1 and NAT2 to highlight their functional similarities and differences. Both NAT1 and NAT2 are potential drug targets for diseases where mitochondrial dysfunction is a hallmark of onset and progression.

Lipidated brush-PEG polymers as low molecular weight pulmonary drug delivery platforms.

OBJECTIVES: Nanomedicines are being actively developed as inhalable drug delivery systems. However, there is a distinct utility in developing smaller polymeric systems that can bind albumin in the lungs. We therefore examined the pulmonary pharmacokinetic behavior of a series of lipidated brush-PEG (5 kDa) polymers conjugated to 1C2, 1C12 lipid or 2C12 lipids. METHODS: The pulmonary pharmacokinetics, patterns of lung clearance and safety of polymers were examined in rats. Permeability through monolayers of primary human alveolar epithelia, small airway epithelia and lung microvascular endothelium were also investigated, along with lung mucus penetration and cell uptake. RESULTS: Polymers showed similar pulmonary pharmacokinetic behavior and patterns of lung clearance, irrespective of lipid molecular weight and albumin binding capacity, with up to 30% of the dose absorbed from the lungs over 24 h. 1C12-PEG showed the greatest safety in the lungs. Based on its larger size, 2C12-PEG also showed the lowest mucus and cell membrane permeability of the three polymers. While albumin had no significant effect on membrane transport, the cell uptake of C12-conjugated PEGs were increased in alveolar epithelial cells. CONCLUSION: Lipidated brush-PEG polymers composed of 1C12 lipid may provide a useful and novel alternative to large nanomaterials as inhalable drug delivery systems.

Utilization of endogenous albumin trafficking pathways in the lungs has potential to modestly increase the lung interstitial access and absorption of drug delivery systems after inhaled administration.

OBJECTIVES: Drug delivery systems typically show limited access to the lung interstitium and absorption after pulmonary delivery. The aim of this work was to undertake a proof-of-concept investigation into the potential of employing endogenous albumin and albumin absorption mechanisms in the lungs to improve lung interstitial access and absorption of inhaled drug delivery systems that bind albumin. METHODS: The permeability of human albumin (HSA) through monolayers of primary human alveolar epithelia, small airway epithelia, and microvascular endothelium were investigated. The pulmonary pharmacokinetics of bovine serum albumin (BSA) was also investigated in efferent caudal mediastinal lymph duct-cannulated sheep after inhaled aerosol administration. RESULTS: Membrane permeability coefficient values (Papp) of HSA increased in the order alveolar epithelia

Dose-Dependent Effect of Phenothiazines as Dynamin II Inhibitors on the Uptake of PEGylated Liposomes by Endocytic Cells and In Vivo Pharmacokinetics of PEGylated Liposomal Doxorubicin in Rats.

Dynamin II (dynII) plays a significant role in the internalization pathways of endocytic cells, by allowing membrane invaginations to "bud off". An important class of dynII inhibitors that are used clinically are phenothiazines, such as prochlorperazine (PCZ). PCZ is an antipsychotic drug but is also currently in clinical trials at higher concentrations as an adjuvant in cancer patients that increases the efficacy of monoclonal antibodies at high intravenous doses. It is unknown, however, whether high-dose dynII inhibitors have the potential to alter the pharmacokinetics of co-administered chemotherapeutic nanomedicines that are largely cleared via the mononuclear phagocyte system. This work therefore sought to investigate the impact of clinically relevant concentrations of phenothiazines, PCZ and thioridazine, on in vitro liposome endocytosis and in vivo liposome pharmacokinetics after PCZ infusion in rats. The uptake of fluorescently labeled PEGylated liposomes into differentiated and undifferentiated THP-1 and RAW246.7 cells, and primary human peripheral white blood cells, was investigated via flow cytometry after co-incubation with dynII inhibitors. The IV pharmacokinetics of PEGylated liposomes were also investigated in rats after a 20 min infusion with PCZ. Phenothiazines and dyngo4a reduced the uptake of PEGylated liposomes by THP-1 and RAW264.7 cells in a concentration-dependent manner in vitro. However, dynII inhibitors did not alter the mean uptake of liposomes by human peripheral white blood cells, but endocytic white cells from some donors exhibited sensitivity to phenothiazine exposure. When a clinically relevant dose of PCZ was co-administered with PEGylated liposomal doxorubicin (Caelyx/Doxil) in rats, the pharmacokinetics and biodistribution of liposomes were unaltered. These data suggest that while clinically relevant doses of dynII inhibitors can inhibit the uptake of liposomes by endocytic cells in vitro, they are unlikely to significantly affect the pharmacokinetics of long-circulating, co-administered liposomes.

Contrasting Effects of Temperature on Human Arylamine N-Acetyltransferase and Acetyl Coenzyme A Hydrolase Activities.

There are two human arylamine N-acetyltransferases (NAT1 and NAT2) that have evolved separately and differ in their substrate specificity and tissue localization. In addition to its acetyltransferase activity, NAT1 can hydrolyze acetyl coenzyme A to coenzyme A in the presence of folate. Here, we show that NAT1 is rapidly inactivated at temperatures above 39 °C whereas NAT2 is more stable. NAT1 acetyltransferase activity is also rapidly lost in whole cells at a rate similar to that of recombinant protein, suggesting it is not protected by intracellular chaperones. By contrast, the hydrolase activity of NAT1 is resistant to heat-induced inactivation, in part because folate stabilizes the protein. Heat generated by mitochondria following the dissipation of the inner membrane potential was sufficient to inactivate NAT1 in whole cells. Within the physiological range of core body temperatures (36.5-37.5 °C), NAT1 acetyltransferase activity decreased by 30% while hydrolase activity increased by >50%. This study demonstrates the thermal regulation of NAT1, but not NAT2, and suggests that NAT1 may switch between an acetyltransferase and a hydrolase within a narrow temperature range in the presence of folate.

Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats.

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 µg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.

Arylamine N-acetyltransferase 1 deficiency inhibits drug-induced cell death in breast cancer cells: switch from cytochrome C-dependent apoptosis to necroptosis.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1) deficiency has been associated with drug resistance and poor outcomes in breast cancer patients. The current study aimed to investigate drug resistance in vitro using normal breast cancer cell lines and NAT1-deficient cell lines to understand the changes induced by the lack of NAT1 that resulted in poor drug response. METHODS: The response to seven chemotherapeutic agents was quantified following NAT1 deletion using CRISPR-Cas 9 in MDA-MB-231 and T-47D cells. Apoptosis was monitored by annexin V staining and caspase 3/7 activity. Cytochrome C release and caspase 8 and 9 activities were measured by Western blots. Caspase 8 was inhibited using Z-IETD-FMK and necroptosis was inhibited using necrostatin and necrosulfonamide. RESULTS: Compared to parental cells, NAT1 depleted cells were resistant to drug treatment. This could be reversed following NAT1 rescue of the NAT1 deleted cells. Release of cytochrome C in response to treatment was decreased in the NAT1 depleted cells, suggesting suppression of the intrinsic apoptotic pathway. In addition, NAT1 knockout resulted in a decrease in caspase 8 activation. Treatment with necrosulfonamide showed that NAT1 deficient cells switched from intrinsic apoptosis to necroptosis when treated with the anti-cancer drug cisplatin. CONCLUSIONS: NAT1 deficiency can switch cell death from apoptosis to necroptosis resulting in decreased response to cytotoxic drugs. The absence of NAT1 in patient tumours may be a useful biomarker for selecting alternative treatments in a subset of breast cancer patients.

Polymorphism in the human arylamine N-acetyltransferase 1 gene 3'-untranslated region determines polyadenylation signal usage.

Human arylamine N-acetyltransferase 1 (NAT1) encodes a drug-metabolising enzyme that plays a role in chemical-associated cancer risk, cancer cell survival and mitochondrial function. Its expression and protein activity are regulated by transcriptional, translational, and post-translational processes, including microRNAs such as miR-1290. Several studies have shown the presence of multiple polyadenylation sites in the NAT1 gene. However, their role in NAT1 expression is poorly understood. Here, we have investigated the genetic sequence of the NAT1 gene in human cell lines, peripheral blood mononuclear cells and breast tumour tissue. We identified five potential polyadenylation signals, two of which carry known single nucleotide polymorphism that affect site usage. Cells that are homozygous for adenine at base 1642, the most distal polyadenylation site, use this site whereas those homozygous for cytosine at base 1642 could not. We also found that the presence of adenine at base 1642 is associated with the NAT1*10 haplotype. Because the putative binding site for miR-1290 is located between the last two polyadenylation sites, we hypothesised that cells that do not use the most distal site will be unaffected by miR-1290. However, this was not the case. NAT1 activity was positively correlated with miR-1290, and induction of miR-1290 in SH-SY5Y cells was associated with induction, not inhibition, of NAT1 activity. The use of PolyA1264 or PolyA1642 did not alter NAT1 activity following ectopic expression of a miR-1290 mimic. These results suggest that the role of miR-1290 in the regulation of NAT1 activity is more complex than previously reported.

Poly(HPMA-co-NIPAM) copolymer as an alternative to polyethylene glycol-based pharmacokinetic modulation of therapeutic proteins.

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.

Monocytes Do Not Contribute to Sex Differences Seen in the Pharmacokinetics of Pegylated Liposomal Doxorubicin.

Pegylated liposomal doxorubicin (PLD) is widely utilised in cancer chemotherapy, but exhibits large inter-individual pharmacokinetic variability and sex differences in plasma clearance. Population pharmacokinetic modelling has suggested PLD plasma clearance correlates with peripheral monocyte counts, while sex hormones are known to affect endocytosis and phagocytosis in mononuclear cells. This study investigated whether sex hormones affect the uptake of PLD by human monocytes and macrophages in vitro. 17ß-estradiol, but not testosterone, inhibited the uptake of PLD in a concentration dependent manner in undifferentiated (but not differentiated) THP-1 cells, and primary monocytes obtained from women, but not men. Effects of estradiol were only evident at very high concentrations seen during pregnancy. No differences were observed in monocyte count or monocyte subtypes between males and females. These data show that monocytes do not contribute to sex differences seen in PLD clearance in humans of reproductive age.

Hypoxia-mediated drug resistance in breast cancers.

Tissue hypoxia in solid tumors is caused by several pathological changes associated with tumor growth, including altered microvasculature structure, increased diffusional distances, and tumor-associated anemia. As the oxygen tension decreases, tumor cells adapt to the limited oxygen supply. Previous studies have shown that such adaptation leads to an aggressive phenotype that is resistant to many anti-cancer therapies. Induction of hypoxia inducible factors (HIFs) mediates many proteomic and genomic changes associated with tumor hypoxia. In breast cancers, HIFs not only predict poor prognosis, but also promote metastasis and drug resistance. Several studies have proposed HIF-1α as a druggable target in drug-resistant breast cancers, leading to the synthesis and development of small molecule inhibitors. Disappointingly, however, none of these small molecule inhibitors have progressed to clinical use. In this review, we briefly discuss the role of HIF-1α in breast cancer drug resistance and summarize the current and future approaches to targeting this transcription factor in breast cancer treatment.

Modulation of Human Arylamine N-Acetyltransferase 1 Activity by Lysine Acetylation: Role of p300/CREB-Binding Protein and Sirtuins 1 and 2.

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.

Interaction of the Brain-Selective Sulfotransferase SULT4A1 with Other Cytosolic Sulfotransferases: Effects on Protein Expression and Function.

Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT: The catalytically inactive sulfotransferase (SULT) 4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.

Effect arylamine N-acetyltransferase 1 on morphology, adhesion, migration, and invasion of MDA-MB-231 cells: role of matrix metalloproteinases and integrin αV.

Reducted arylamine N-acetyltransferase (NAT1) in breast cancers is associated with poor patient survival. NAT1 has also been associated with changes in cancer cell survival and invasion both invitro and invivo. Here, we report the effects of NAT1 in cancer cell invasion by addressing its role in adherence, migration, and invasion in vitro. The NAT1 gene was deleted in MDA-MB-231, HT-29 and HeLa cells using CRISPR/Cas9 gene editing. Loss of NAT1 increased adherence to collagen in all three cell-lines but migration was unaffected. NAT1 deletion decreased invasion and induced changes to cell morphology. These effects were independent of matrix metalloproteinases but were related to integrin ITGαV expression. The data suggest NAT1 is important in adhesion and invasion through integrin expression.

Arylamine N-Acetyltransferase 1 Regulates Expression of Matrix Metalloproteinase 9 in Breast Cancer Cells: Role of Hypoxia-Inducible Factor 1-α.

Arylamine N-acetyltransferase 1 (NAT1) is a drug-metabolizing enzyme that influences cancer cell proliferation and survival. However, the mechanism for these effects is unknown. Because of previous observations that NAT1 inhibition decreases invasiveness, we investigated the expression of the metalloproteinase matrix metalloproteinase 9 (MMP9) in human breast cancer samples and in cancer cells. We found a negative correlation between the expression of NAT1 and MMP9 in 1904 breast cancer samples. Moreover, when NAT1 was deleted in highly invasive breast cancer cells, MMP9 mRNA and protein significantly increased, both of which were reversed by reintroducing NAT1 into the knockout cells. After NAT1 deletion, there was an increased association of acetylated histone H3 with the SET and MYND-domain containing 3 (SMYD3) element in the MMP9 promoter, consistent with an increase in MMP9 transcription. NAT1 deletion also up-regulated hypoxia-inducible factor 1-α (HIF1-α). Treatment of the NAT1 knockout cells with small interfering RNA directed toward HIF1-α mRNA inhibited the increased expression of MMP9. Taken together, these results show a direct inverse relationship between NAT1 and MMP9 and suggest that HIF1-α may be essential for the regulation of MMP9 expression by NAT1. SIGNIFICANCE STATEMENT: The expression of the enzyme NAT1 was found to be negatively correlated with MMP9 expression in tumor tissue from breast cancer patients. In cells, NAT1 regulated MMP9 expression at a transcriptional level via HIF1-α. This finding is important as it may explain some of the pathological features associated with changes in NAT1 expression in cancer.

Loss of human arylamine N-acetyltransferase I regulates mitochondrial function by inhibition of the pyruvate dehydrogenase complex.

Human arylamine N-acetyltransferase 1 (NAT1) has been widely reported to affect cancer cell growth and survival and recent studies suggest it may alter cell metabolism. In this study, the effects of NAT1 deletion on mitochondrial function was examined in 2 human cell lines, breast carcinoma MDA-MB-231 and colon carcinoma HT-29 cells. Using a Seahorse XFe96 Flux Analyzer, NAT1 deletion was shown to decrease oxidative phosphorylation with a significant loss in respiratory reserve capacity in both cell lines. There also was a decrease in glycolysis without a change in glucose uptake. The changes in mitochondrial function was due to a decrease in pyruvate dehydrogenase activity, which could be reversed with the pyruvate dehydrogenase kinase inhibitor dichloroacetate. In the MDA-MB-231 and HT-29 cells, pyruvate dehydrogenase activity was attenuated either by an increase in phosphorylation or a decrease in total protein expression. These results may help explain some of the cellular events that have been reported recently in cell and animal models of NAT1 deficiency.

A 30 kDa polyethylene glycol-enfuvirtide complex enhances the exposure of enfuvirtide in lymphatic viral reservoirs in rats.

HIV therapy with anti-retroviral drugs is limited by the poor exposure of viral reservoirs, such as lymphoid tissue, to these small molecule drugs. We therefore investigated the effect of PEGylation on the anti-retroviral activity and subcutaneous lymphatic pharmacokinetics of the peptide-based fusion inhibitor enfuvirtide in thoracic lymph duct cannulated rats. Both the peptide and the PEG were quantified in plasma and lymph via ELISA. Conjugation to a single 5 kDa linear PEG decreased anti-HIV activity three-fold compared to enfuvirtide. Whilst plasma and lymphatic exposure to peptide mass was moderately increased, the loss of anti-viral activity led to an overall decrease in exposure to enfuvirtide activity. A 20 kDa 4-arm branched PEG conjugated with an average of two enfuvirtide peptides decreased peptide activity by six-fold. Plasma and lymph exposure to enfuvirtide, however, increased significantly such that anti-viral activity was increased two- and six-fold respectively. The results suggest that a multi-enfuvirtide-PEG complex may optimally enhance the anti-retroviral activity of the peptide in plasma and lymph.

The MBNL/CELF Splicing Factors Regulate Cytosolic Sulfotransferase 4A1 Protein Expression during Cell Differentiation.

Sulfotransferase 4A1 (SULT4A1) is a sulfotransferase-like protein that is highly conserved between species. In human tissues, there are two transcripts, one that produces a full-length protein and one that produces an unstable truncated protein. The second transcript, which includes a pseudo-exon between exons 6 and 7 (6p), is widely expressed, whereas the first is more restricted. Differentiation of neuronal cells results in the removal of the pseudo-exon and subsequent SULT4A1 protein expression. Recent studies with SULT4A1 knockout mice showed that the protein is essential for normal development and that its absence leads to a severe neurologic phenotype. Here, the regulation of SULT4A1 6p splicing was investigated during neuronal differentiation using SH-SY5Y cells, human induced pluripotent stem cells, and mouse embryonic tissue. In all three models, pseudo-exon 6p was removed during differentiation, resulting in stable SULT4A1 protein expression. Using a minigene splicing assay, a region upstream of pseudo-exon 6p was identified that is essential for correct splicing of SULT4A1 mRNA. Within this region, there were binding motifs for four RNA processing factors (MBNL-1, MBNL-2, CELF-1, and CELF-2). Time-dependent changes in SULT4A1 protein and MBNL/CELF protein during differentiation supported their role in correctly splicing the SULT4A1 mRNA. Furthermore, ectopic expression of each factor produced efficient splicing in the minigene assay as well as correct splicing of the endogenous SULT4A1 mRNA. These results show that SULT4A1 mRNA is a target for MBNL/CELF-dependent splicing, which may be essential in producing stable, functional SULT4A1.