Alkylated Imino Sugars Research Articles

Alkyl-imino sugars inhibit the pro-oncogenic ion channel function of human papillomavirus (HPV) E5

Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus-coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5-mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK-MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high-risk HPV types. Moreover, viroporin inhibitors also reduced ERK-MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high-risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation.

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher KI) and a lower rate of enzyme inactivation (k(inact)). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5-6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.

Pharmaceutical Approaches and Advancements in Male Contraception

Currently available contraceptive methods offer a variety of options for women, but only very few for men which include surgical methods, condom and hormonal methods. Non-surgical and non-hormonal methods are under investigation. Among these, hormonal contraceptive approaches, including injections, oral and transdermal delivery systems of testosterone, have attracted the attention of investigators. Also non-hormonal approaches based on chemicals extracted from different plants such as cotton seed plant, Neem tree, Trypterigium wilfordii and Momordica Charantia seed, are known to have effect on male fertility. Additionally, alkylated imino sugars, Ca ++ channel blockers, indenopyridines, indazole-3-carboxylic acid analogues, reversible inhibition of sperm under guidance (RISUG) which involves injection of stericmaleic anhydride with dimethyl sulfoxide, spermicide‐ microbicide (including gel formulations) and vaccine approaches are intended to interfere in a certain fertilization step. Information obtained from multi-center studies in several countries on both men or women shows the necessity for additional reversible male contraceptive methods. Results from recent surveys clearly indicate that there is a market and a need for novel pharmaceutical preparations for male contraception.

Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel

The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.

Accumulation of Glucosylceramide in Murine Testis, Caused by Inhibition of β-Glucosidase 2: IMPLICATIONS FOR SPERMATOGENESIS

One of the hallmarks of male germ cell development is the formation of a specialized secretory organelle, the acrosome. This process can be pharmacologically disturbed in C57BL/6 mice, and thus infertility can be induced, by small molecular sugar-like compounds (alkylated imino sugars). Here the biochemical basis of this effect has been investigated. Our findings suggest that in vivo alkylated imino sugars primarily interact with the non-lysosomal glucosylceramidase. This enzyme cleaves glucosylceramide into glucose and ceramide, is sensitive to imino sugars in vitro, and has been characterized as beta-glucosidase 2 (GBA2). Imino sugars raised the level of glucosylceramide in brain, spleen, and testis, in a dose-dependent fashion. In testis, multiple species of glucosylceramide were similarly elevated, those having long acyl chains (C16-24), as well as those with very long polyunsaturated acyl chains (C28-30:5). Both of these GlcCer species were also increased in the testes from GBA2-deficient mice. When considering that the very long polyunsaturated sphingolipids are restricted to germ cells, these results indicate that in the testis GBA2 is present in both somatic and germ cells. Furthermore, in all mouse strains tested imino sugar treatment caused a rise in testicular glucosylceramide, even in a number of strains, of which the males remain fertile after drug administration. Therefore, it appears that acrosome formation can be derailed by accumulation of glucosylceramide in an extralysosomal localization, and that the sensitivity of male germ cells to glucosylceramide is genetically determined.

The postacrosomal assembly of sperm head protein, PAWP, is independent of acrosome formation and dependent on microtubular manchette transport

PAWP (postacrosomal sheath WW domain-binding protein) exclusively resides in the postacrosomal sheath (PAS) of the sperm perinuclear theca (PT). Because of the importance of this region in initiating oocyte activation during mammalian fertilization [Sutovsky, P., Manandhar, G., Wu, A., Oko, R., 2003. Interactions of sperm perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction. Microsc. Res. Tech. 61, 362–378; Wu, A., Sutovsky, P., Manandhar, G., Xu, W., Katayama, M., Day, B.N., Park, K.W., Yi, Y.J., Xi, Y.W., Prather, R.S., Oko, R., 2007. PAWP, A sperm specific ww-domain binding protein, promotes meiotic resumption and pronuclear development during fertilization. J. Biol. Chem. 282, 12164–12175], we were interested in resolving the origin and assembly of its proteins during spermatogenesis, utilizing PAWP as a model. Based on previous PT developmental studies, we predicted that the assembly of PAWP is dependent on microtubule-manchette protein transport and manchette descent and independent of subacrosomal PT formation. Consequently, we hypothesized that PAWP will colocalize with manchette microtubules during spermiogenesis. Utilizing specific antibodies, PAWP was first detected in the cytoplasmic lobe of spermatids beginning to undergo elongation and became most prominent in this region just prior to and during manchette descent. During this peak period, PAWP was concentrated over the manchette and colocalized with alpha- and beta-tubulin. It was then assembled as part of the PAS in the wake of manchette descent over the caudal half of the elongated spermatid nucleus. PAWP mRNA, on the other hand, was first detected in mid-pachytene spermatocytes, peaked by early round spermatids, and declined during spermatid elongation. In order to confirm that PAWP-PAS assembly was independent of subacrosomal PT development, PAWP immunolocalization was performed on the testes of NB-DNJ-treated mice which fail to form an acrosome and subacrosomal layer during spermiogenesis [van der Spoel, A.C., Jeyakumar, M., Butters, T.D., Charlton, H.M., Moore, H.D., Dwek, R.A., Platt, F.M., 2002. Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception. Proc. Natl. Acad. Sci. U.S.A. 99, 17173–17178] but whose elongated spermatids still retain egg-activating ability [Suganuma, R., Walden, C.M., Butters, T.D., Platt, F.M., Dwek, R.A., Yanagimachi, R., and van der Spoel, A.C., 2005. Alkylated imino sugars, reversible male infertility-inducing agents, do not affect the genetic integrity of male mouse germ cells during short-term treatment despite induction of sperm deformities. Biol. Reprod. 72, 805–813]. The same temporal and manchette-based pattern of PAWP-PAS assembly during spermiogenesis was evident as in controls supporting our hypothesis that PAS assembly is independent of subacrosomal PT formation and that egg-activating ability resides within the PAS.

Alkylated Imino Sugars, Reversible Male Infertility-Inducing Agents, Do Not Affect the Genetic Integrity of Male Mouse Germ Cells During Short-Term Treatment Despite Induction of Sperm Deformities1

Reversible infertility can be induced in male mice by oral administration of the alkylated imino sugars N-butyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ). Spermatozoa of these mice have grossly misshapen heads and reduced motility. Because NB-DNJ and related compounds may hold promise as nonhormonal male contraceptives, a comprehensive examination of their effects on male reproduction is necessary. To this end, we further examined reproductive properties of the dysmorphic spermatozoa that are produced after short-term imino sugar administration at the minimal dose that completely abolishes the ability of male C57BL/6 mice to produce offspring by natural mating. Here, we report that, in vitro, the abnormal spermatozoa from the NB-DNJ- and NB-DGJ-treated mice were unable to fertilize oocytes. In addition, we investigated whether the imino sugars damage the genetic integrity of spermatozoa. To test this, we microsurgically injected deformed spermatozoa from imino sugar-treated males into oocytes. The deformed spermatozoa from the testis were able to activate oocytes very efficiently, but those from the cauda epididymis often failed to do so. This problem was overcome when the sperm-injected oocytes were treated with a parthenogenetic agent, Sr(2+). Oocytes injected with the misshapen spermatozoa from NB-DNJ- and NB-DGJ-treated mice developed (with or without Sr(2+) treatment) into live offspring that grew normally and were normally fertile. This indicates that during short-term administration, alkylated imino sugars alter sperm morphology and physiology but do not diminish the genetic potential of spermatozoa.

Reversible infertility in male mice after oral administration of alkylated imino sugars: a nonhormonal approach to male contraception.

During mammalian spermatogenesis, male germ cells undergo a dramatic transformation, which includes a change of shape, nuclear condensation, and development of specialised structures, such as an acrosome, and a flagellum with a mitochondrial sheath. We have found a previously undescribed pharmacological approach to intervene in these events. After oral administration of the alkylated imino sugar N-butyldeoxynojirimycin (NB-DNJ) to mice, epididymal spermatozoa displayed a spectrum of abnormal head shapes, and acrosomal antigens were mostly absent or displayed irregular patterns. In addition, the mitochondria of these cells often had an aberrant morphology, and were arranged in relatively short and wide mitochondrial sheaths. The motility of the affected spermatozoa was severely impaired. After 3 weeks of administration of NB-DNJ, male mice became sterile, and regained their fertility during the fourth week off drug. The NB-DNJ-induced infertility was not associated with a reduction in the serum testosterone level. Biochemically, the capacity of imino sugars to impair spermatogenesis was associated with their potential to attenuate the biosynthesis of glucosylceramide-based sphingolipids. Our study reveals that male fertility is affected by partial glycosphingolipid depletion, or, alternatively, by a distinct as yet unidentified property that is shared by alkylated imino sugars that inhibit glucosylceramide biosynthesis. These compounds therefore may be new leads in the development of a male contraceptive, especially because NB-DNJ has already been through extensive evaluation in various mammals, including man.

Preparation, biochemical characterization and biological properties of radiolabelled N-alkylated deoxynojirimycins.

We have reductively alkylated deoxynojirimycin imino sugars using sodium cyanoborohydride to provide an efficient means of generating a series of N-alkylated compounds containing 4-18 carbon side chains. The yields were greater than 90% using a variety of aldehydes of different chain lengths, and after purification were >95% pure using (1)H-NMR. Radiolabelled compounds were prepared using sodium cyanoborotriti-ide to selectively label the first carbon atom in the alkyl chain and used in protein-binding and cell- and tissue-uptake experiments. Protein binding was chain-length-dependent with compounds of intermediate chain length (C(9)-C(12)), demonstrating an equal distribution between the aqueous and protein-bound phase. The extent of cell uptake also increased proportionally with increased chain length in a time-dependent manner. When administered to mice, the longer alkyl-chain compounds showed reduced absorption from the intestine and a marked deposition of compound in the liver and brain, suggesting that the more hydrophobic compounds were poorly cleared by the major tissues. In tissue-culture cells compounds with 8 or fewer carbon atoms were non-toxic and had CC(50) (the concentration at which the number of cells or cell proliferation is reduced by 50%) values greater than 1 mM. Compounds with chain lengths above C(8) showed a chain-length-dependent increase in cytotoxicity. N-alkylated deoxynojirimycins (C(4)-C(18)) were evaluated for their inhibitory effects on ceramide-specific glucosyltransferase and glycoprotein-processing alpha-glucosidase. Increasing the alkyl chain length had little effect on alpha-glucosidase activity, but inhibition of ceramide-specific glucosyltransferase increased 10-fold when C(4) and C(9)-C(18) compounds were compared. Overall these data provide further definition of the molecular features of alkylated imino sugars that influence tissue selectivity and efficacy for cellular enzyme inhibition.

Iminosugars as possible broad spectrum anti hepatitis virus agents: the glucovirs and alkovirs.

Glucosidases in the endoplasmic reticulum (ER) mediate the first step in processing N-linked oligosaccharides. Recent evidence suggests that morphogenesis and secretion of members of the hepatitis B and flavivirus families are more dependent on these enzymes than are most host glycoproteins. Thus, it is possible that glucosidase inhibitors can be designed that are safe and selective for the treatment of hepatitis B and possibly C (since hepatitis C virus is a member of the flavivirus family), making them broad spectrum with respect to hepatitis viruses. Numerous pharmacological and genetic dissections support the notion that glucosidase inhibition can have an antiviral effect, and imino sugars that competitively inhibit ER glucosidases have been proposed as anti-hepatitis drug candidates. We call this family of compounds 'glucovirs'. Recently, however, alkylated imino sugars that retain substantial antiviral activity but lack glucosidase inhibitory activity have been described. These compounds are called 'alkovirs' and their mechanism of action is unknown. This review considers the rationale of the glucovir and alkovir approach to the treatment of hepatitis B and C.