Abstract
5α-Reduced glucocorticoids (GCs) are formed when one of the two isozymes of 5α-reductase reduces the Δ4–5 double bond in the A-ring of GCs. These steroids are largely viewed inert, despite the acceptance that other 5α-dihydro steroids, e.g. 5α-dihydrotestosterone, retain or have increased activity at their cognate receptors. However, recent findings suggest that 5α-reduced metabolites of corticosterone have dissociated actions on GC receptors (GRs) in vivo and in vitro and are thus potential candidates for safer anti-inflammatory steroids. 5α-Dihydro- and 5α-tetrahydro-corticosterone can bind with GRs, but interest in these compounds had been limited, since they only weakly activated metabolic gene transcription. However, a greater understanding of the signalling mechanisms has revealed that transactivation represents only one mode of signalling via the GR and recently the abilities of 5α-reduced GCs to suppress inflammation have been demonstrated in vitro and in vivo. Thus, the balance of parent GC and its 5α-reduced metabolite may critically affect the profile of GR signalling. 5α-Reduction of GCs is up-regulated in liver in metabolic disease and may represent a pathway that protects from both GC-induced fuel dyshomeostasis and concomitant inflammatory insult. Therefore, 5α-reduced steroids provide hope for drug development, but may also act as biomarkers of the inflammatory status of the liver in metabolic disease. With these proposals in mind, careful attention must be paid to the possible adverse metabolic effects of 5α-reductase inhibitors, drugs that are commonly administered long term for the treatment of benign prostatic hyperplasia.
Introduction
Glucocorticoids (GCs) are steroid hormones synthesised in the adrenal cortex, which exert a plethora of effects in the body, ranging from modulation of metabolism and suppression of inflammation to regulation of stress responses and neuronal function. These hormones are vital for health and this is most vividly demonstrated in disorders characterised by GC deficiency or excess. Diminished GC production, as seen in Addison's disease, leads to critical illness and is life-threatening if inadequately treated. The converse is seen in Cushing's syndrome, where excess GC production manifests as insulin resistance, obesity, dyslipidaemia, altered immune responses (e.g. impaired wound healing) and disturbed central nervous system activity (e.g. depression).
While endogenous GCs play a key role in many aspects of health and disease, synthetic exogenous GCs have become widely used therapeutically, predominantly as anti-inflammatory agents. Many synthetic GC variants have been developed, aiming to maximise their clinical utility through optimisation of pharmacological properties. In the UK, it is estimated that ∼40 million prescriptions for GCs are dispensed each year, with ∼70% of these being either inhaled or topical preparations (http://www.isd.scot.nhs.uk/isd/1038.html, http://www.ic.nhs.uk/statistics-and-data-collections/primary-care/prescriptions). Despite their success in treating inflammatory conditions, synthetic GCs remain plagued by side effects wrought through widespread activation of the ubiquitous GC receptors (GRs). Receiving GCs therapeutically incurs a dose-dependent rise in the relative risk of cardiovascular morbidity (Souverein et al. 2004, Wei et al. 2004, Solomon et al. 2011), with an increase of up to threefold amongst current users of high-dose GCs. The relative risk for any fracture in those using GCs is 1.33, and this increase in risk is particularly marked for vertebral fractures with a relative risk of 2.60 (Canalis et al. 2007, van Staa et al. 2011). On this background, there is considerable interest in designing GR ligands that have the ability to suppress inflammation without modulating metabolism or adversely affecting bone remodelling. However, it has been difficult to achieve this goal.
Therapeutic actions of GCs to relieve inflammation
GCs mediate many of their effects by binding with the GR (NR3C1; Hollenberg et al. 1985), a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. The conventional model (Beato et al. 1995) of GR action is of GR homodimers modulating gene transcription through direct interactions with DNA. In the unbound state, monomeric GR is located in the cytosol in association with a complex of chaperone proteins. Ligand binding induces conformational changes resulting in dissociation of the chaperone complex, in turn enabling exposure of the DNA binding and dimerisation domains and permitting nuclear translocation. A GR homodimer interacts with GC response elements (GREs) within the promoter region of target genes in the nucleus, leading to up-regulation of gene transcription (transactivation). Multiple genes encoding critical proteins in metabolic pathways, such as tyrosine aminotransferase (TAT; Grange et al. 2001) or phosphoenolpyruvate carboxykinase (PEPCK; Petersen et al. 1988, Ruppert et al. 1990), are up-regulated in this manner. This mechanism of transactivation underpins many of the metabolic side effects of these drugs. In contrast, GR can also down-regulate gene transcription by binding with negative GRE sites. Examples of genes regulated in this way are POMC (Drouin et al. 1993) and osteocalcin (Meyer et al. 1997b), where GR multimers bind to negative GREs within promoter regions, repressing gene transcription and influencing negative feedback on the hypothalamic–pituitary–adrenal (HPA) axis and bone remodelling respectively.
However, not all responses to GCs are mediated in a manner solely dependent on GR dimers binding with DNA, and indeed, the ability of GCs to suppress inflammation involves many other modalities of receptor signalling. Those pro-inflammatory signalling pathways believed to be most important in GC action have been reviewed extensively (De Bosscher & Haegeman 2009) and are summarised in Fig. 1. GR monomers play a crucial role in the repression of pro-inflammatory genes. These actions are attributed to GR monomers tethering with DNA-bound pro-inflammatory transcription factors NF-κB and AP-1, thus preventing their actions activating transcription of pro-inflammatory cytokines such as TNFα and IL6 (Barnes 1998). This mechanism is distinct from those described above as it requires neither GR dimerisation nor direct DNA binding of the GR monomer. The relative importance of such dimerisation-independent mechanisms to suppress inflammation was elegantly demonstrated in GRdim/dim mice, which express a mutant GR incapable of dimerisation (Reichardt et al. 2001). These mice were still able to respond to dexamethasone, suppressing expression of pro-inflammatory cytokines.
Glucocorticoid (GC) effects on inflammatory signalling. GCs act through several mechanisms to exert anti-inflammatory effects: 1) non-genomic pathways involve GC receptor (GR)-mediated direct interactions with second messenger proteins, including the MAPK protein JNK, inhibiting the activation of this signalling pathway. 2) GR-mediated transactivation of key anti-inflammatory genes involves direct DNA binding of both GR dimers and monomers/multimers to GC-response elements (GRE) in the promoter region of target gene. 3) Transrepression of pro-inflammatory genes does not require direct DNA binding of GR, but rather ‘tethering’ of GR monomers to DNA-bound pro-inflammatory transcription factors.
Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0318
Whilst the predominant anti-inflammatory actions of GCs are mediated through GR monomer-based repression of pro-inflammatory transcription factor activity, some of the actions of GR to reduce inflammation are mediated through transactivation mechanisms. Here, GR DNA binding is essential, but dimerisation is not always required. For example, transcription of the anti-inflammatory protein IκBα results from GR dimers interacting with hormone response elements located within its promoter region (Heck et al. 1997, Deroo & Archer 2001). However, transactivation of other anti-inflammatory genes including activation of interleukin 10 (IL10) have been shown to occur independently of dimerisation (Unterberger et al. 2008). This has also been demonstrated for MKP1 (also known as DUSP1) gene expression, which is induced by GC treatment even in the presence of the dimerisation-deficient GR mutant (Abraham et al. 2006). Since prevention of dimerisation does not abrogate all of the anti-inflammatory actions of GR, this gives hope that the independent mechanisms underlying these pathways may be tractable to pharmaceutical manipulation and aid rational design of new drugs. Simply dissociating transrepression from transactivation may not represent the best approach (Belvisi et al. 2001) and indeed to achieve tissue-specific selectivity, targeting specific interactions between GR and co-activator/co-repressor proteins may be more successful (Coghlan et al. 2011).
There have been numerous attempts to develop ‘dissociated’ steroids or ‘selective GR modulators’ with improved therapeutic indices (McMaster & Ray 2008). This strategy has been successful previously in the development of selective oestrogen receptor modulators, where careful manipulation of intracellular interactions of nuclear hormone receptors with co-regulators or co-repressors has led to selectivity of actions (oestrogenic effects on reproductive tissues vs bone; Smith & O'Malley 2004). Endogenous steroids have been utilised as templates in the pharmaceutical development of synthetic GCs. In humans, the most abundant, naturally occurring GC is cortisol (Fig. 2), whereas in rodents (lacking adrenal 17α-hydroxylase), corticosterone is the active species formed (Fig. 2). Corticosterone is also produced in humans but at a rate approximately one-seventh of that of cortisol (Van der Straeten et al. 1963), achieving circulating levels ∼10–30 times lower (Mattingly et al. 1989, Hariharan et al. 1992, Ghulam et al. 1999). These steroids are viewed as the only endogenous GCs, binding tightly to the GR with Kds of ∼3.5 and 10–30 nM respectively (Reul et al. 1990, Mulatero et al. 1997). However, they are not specific for these receptors and can also bind with other nuclear hormone receptors with high affinity, for example to MR (Kds similar to that of aldosterone, ∼1.3 nM (Arriza et al. 1987)). Corticosterone can interact with the pregnane X receptor (PXR) or rodent steroid X receptor (SXR) to induce transcription but activates this less potently than GR (Blumberg et al. 1998).
Metabolism of cortisol and corticosterone to generate 5α-reduced glucocorticoids. Generic scheme of reaction catalysed by 5α-reductases (5αRs) followed by 3α-hydroxysteroid dehydrogenases (3αHSDs). Specific reactions relating to cortisol, corticosterone and testosterone are shown.
Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0318
Synthetic GCs used clinically have structural modifications aimed at enhancing binding affinity with, and specificity for, GR. As with endogenous GCs, synthetic derivatives have varying degrees of action at other receptors, particularly MR. Dexamethasone has a Kd for GR of ∼5 nM (Ray et al. 1999), but lacks significant agonist activity at MR and is devoid of adverse effects on electrolyte balance. While prednisolone strongly binds GR, it also has affinity for MR (Coghlan et al. 2011), thus retaining some mineralocorticoid activity. All synthetic GCs commonly used in clinical practice have potential metabolic and osteopenic side effects, since they were not designed to discriminate between the various facets of GR actions. A novel drug that successfully discriminates side effects from anti-inflammatory actions has not been established in clinical use. Indeed, although some earlier candidates did make it to the market, e.g. deflazacort, when dose equivalency was fully checked, they still possessed an adverse side effect profile (Markham & Bryson 1995). However, some agents have come to the fore in more recent pre-clinical studies, which validate the potential of the approach (De Bosscher & Haegeman 2009), e.g. compound A, a natural product derived from a Namibian desert plant (De Bosscher et al. 2011) and AL-438 (Coghlan et al. 2011) developed by Abbott. Although they bind selectively with GR, not all of these novel agents are steroidal in nature.
5α-Reduced GCs: the case for natural selection?
Recently, the anti-inflammatory potential of some endogenous metabolites of corticosterone, namely 5α-dihydro and 5α-tetrahydro-corticosterone (Fig. 2) have been investigated (McInnes et al. 2004, Yang et al. 2011). Endogenous GCs (cortisol and corticosterone) are principally inactivated in the liver, yielding polar metabolites that can be more rapidly cleared by the kidneys. The main routes of clearance are via A-ring reduction yielding two stereoisomeric metabolites, 5α- and 5β-tetrahydro steroids, following sequential reduction of the Δ4–5 double bond and then the 3-ketone group (Andrew & Walker 2005). 5α-Reductases were initially described and characterised in the 1950s and 1960s stimulated by work in rodents revealing the presence of hepatic enzymes that catalysed 5α-reduction of steroids (Schneider & Horstmann 1951, Schneider 1952, Forchielli & Dorfman 1956). Although the presence of multiple enzymes was initially suggested (McGuire & Tomkins 1960, McGuire et al. 1960, Godoy et al. 2011), genes encoding two isozymes (SRD5A1 and SRD5A2) were later cloned in the 1990s (Andersson & Russell 1990, Andersson et al. 1991).
SRD5A1 and SRD5A2 are members of a larger family of genes containing 5α-reductase domains, the other members being encoded by the genes SRD5A3 and GPSN2-SRD5A2L2, whose roles remain less clear, but may involve reduction of non-steroidal substrates (Cantagrel et al. 2010). SRD5A3 was identified through expression profiling of hormone refractory prostate cancer cells and its transcript is expressed in higher levels in malignancy in comparison with benign tissues (Uemura et al. 2008, Yamana et al. 2010, Godoy et al. 2011). Conservation in the C- and N-terminal regions between it and 5α-reductases 1 and 2 (5αR1 and 5αR2) supports a role for resultant SRD5A3 protein in the metabolism of steroids, a hypothesis was borne out using constructs expressing a protein capable of metabolising testosterone (Uemura et al. 2008) and androstenedione (Yamana et al. 2010) to their 5α-dihydro metabolites. To date, similar catalytic activities have not been demonstrated for GCs. However, a distinct role for this enzyme has been proposed to catalyse conversion of polyprenol to dolichol and mutations in SRD5A3 give rise to a congenital disorder of glycosylation (Cantagrel et al. 2010, Morava et al. 2010, Kahrizi et al. 2011).
5β-Reductase, similarly cloned in the 1990s (Kondo et al. 1994), is a distinct enzyme and part of the aldo-keto reductase superfamily (AKR1D), being most highly expressed in liver (Kimura et al. 1998). It is best recognised for its vital role in bile acid synthesis (Kimura et al. 1998). Both configurations of A-ring-reduced metabolites of GCs were generally considered inert. However, recently, 5α-reduced metabolites of corticosterone have demonstrated attractive pharmaceutical properties in vivo, possessing anti-inflammatory properties, with an apparent paucity of adverse metabolic side effects (Yang et al. 2011). Although not the topic of this review, 5β-reduced GCs also possess some biological activities, having the ability to bind and activate the steroid (or pregnane) X receptor (Blumberg et al. 1998) and can lower intra-ocular pressure (Weinstein et al. 1983, Southren et al. 1985, 1986, 1987) through an uncharacterised mechanism.
Biosynthesis of 5α-reduced GCs
5α-Reduced GCs are formed by 5αR1 and 5αR2 (Andersson & Russell 1990, Andersson et al. 1991). Both isozymes have five exons and four introns and retain a high degree of homology, suggestive of a common origin (Russell & Wilson 1994). There is also an apparently non-functioning pseudogene mapped to the X chromosome (Jenkins et al. 1991). The SRD5As have received most attention for their critical role in the development of the male reproductive system. 5α-Reduced metabolites of testosterone and androstenedione, 5α-dihydrotestosterone or 5α-androstane-3-dione, respectively, possess higher affinity for the androgen receptor (AR) compared with the parent steroid (Wilson & French 1976, Chang et al. 1988, Lubahn et al. 1988). Identification and characterisation of these genes, therefore, led to an understanding of the aetiology of a condition termed pseudohermaphroditism, caused by lack of 5α-reductase 2 (Imperato-McGinley et al. 1974, Walsh et al. 1974, Fisher et al. 1978). This condition, now termed 5α-reductase 2 deficiency, occurs in individuals with 46 XY disorder of sexual development and is characterised by lack of, or reduction in, virilisation at birth, followed by the development of a male phenotype at puberty, though without associated prostatic growth. Polymorphisms in the type 2 gene may also influence the severity and predisposition to prostate cancer, although a recent meta-analysis argues against their importance in this disease (Li et al. 2011). Although the first cDNA clone of 5αR was the type 1 isozyme, a specific and unique role for this enzyme has not been recognised (Andersson & Russell 1990).
Disease states associated with inactivating mutations in 5αR1 have not been identified, although polymorphisms have been reported in polycystic ovarian syndrome (PCOS; Eminovic et al. 2005). One study implicates single nucleotide polymorphisms in both 5αR genes; a variant in SRD5A2 (which encodes a protein with less activity) is protective against PCOS, and several different variants in SRD5A1 are associated with the presence, and increased severity, of hirsutism (Goodarzi et al. 2006). However, others have failed to demonstrate relationships between genetic polymorphisms in SRD5A1 and severity of either hyperandrogenic states in women (Eminovic et al. 2005) or androgenetic alopecia in men (Ha et al. 2003).
5α-Reductases are hydrophobic, membrane-bound enzymes with predicted molecular weights of ∼28–29 kDa (Andersson & Russell 1990). Human 5αR1 and 5αR2 contain 259 (Andersson & Russell 1990) and 254 (Andersson et al. 1991) amino acids respectively. The two isozymes of 5α-reductase have species-specific tissue distributions (summarised for human and rodents in Table 1), which in turn may have implications for formation and action of 5α-reduced GCs. For example, in the immune system, the type 1 isozyme is more widely expressed than type 2, although there are species differences (Milewich et al. 1983, Araneo et al. 1991, Normington & Russell 1992, Zhou et al. 1998, Borlak et al. 2004, Hammer et al. 2005, Samy et al. 2001); Yang et al. (2011) demonstrated an absence of transcripts of both isozymes in bone marrow-derived murine macrophages, compared with previous findings reporting the presence of enzyme in human and rat (Table 1). Activity and/or expression of 5αRs has also been demonstrated in the skin (Dube et al. 1975, Takayasu et al. 1980, Randall et al. 1982, Luu-The et al. 1994, Eicheler et al. 1995, 1998, Sawaya & Price 1997, Thiboutot et al. 2000, Thiele et al. 2005), lungs (Normington & Russell 1992, Provost et al. 2002, Kimura et al. 2003), and in the gastrointestinal tract (Nienstedt et al. 1980a,b, Normington & Russell 1992), all sites of importance in inflammatory diseases and GC therapies. In metabolic tissues, 5αR1 is highly expressed in the liver (Normington & Russell 1992, Livingstone et al. 2005, 2009) but is also present in lower abundance in adipose (Livingstone et al. 2005, 2009, Wake et al. 2007), but not in human skeletal muscle (Thigpen et al. 1993b). 5αR2 is more prevalent in reproductive tissues, although it is present in human liver (Thigpen et al. 1993b) and adipose (MacKenzie et al. 2008). Both isozymes have been reported in primary osteoblasts (Issa et al. 2002), though the significance of 5αRs in bone is not well understood at present. Within the brain, 5αR1 is more highly expressed than 5αR2 (Melcangi et al. 1993, 1998, Poletti et al. 1997a,b, Stoffel-Wagner et al. 1998, 2000, Steckelbroeck et al. 2001, Torres & Ortega 2006), particularly in the white matter, but notably also in the hypothalamus (Karolczak et al. 1998) and pituitary (Lephart 1993, Lephart & Husmann 1993, Lopes-Solache et al. 1996, Yokoi et al. 1996), key sites where GCs exert negative feedback on the HPA axis. It is conceptually possible that manipulation of, or abnormalities in, 5αRs may affect central GC feedback and regulation.
Tissue distribution of 5α reductases in human, rat and mouse. In cases of differing evidence, those demonstrating isozyme-specific expression are shown
| 5αR1 | 5αR2 | Isozyme unconfirmed | ||||||
|---|---|---|---|---|---|---|---|---|
| mRNA | Protein/IC | Activitya | mRNA | Protein/IC | Activitya | Activityb | References | |
| System | ||||||||
| Metabolic/cardiorespiratory | ||||||||
| Liver | H✓; R✓; M✓ | H✓; R✓ | R✓ | H✓; R×; M✓ | H✓; R× | H✓ | 22, 53, 65, 40, 32, 70 | |
| Adipose | H✓; R✓; M✓ | H✓; M× | R✓ | 91, 13, 5, 83, 38, 33 | ||||
| Skeletal muscle | H×; R✓ | H× | H× | H×; R× | H× | H× | 78, 65 | |
| Heart/vessels | R× | R× | H✓; R✓ | 48, 53, 65, 7 | ||||
| Kidney | H×; R✓ | H× | H✓ | H×; R✓ | H× | H✓ | M± | 51, 78, 65 |
| Adrenal | H×; R✓; M± | H×; R✓ | H✓ | H×; R✓; M✓ | H× | H× | R✓ | 10, 78, 65, 89, 37 |
| Lung | H✓; R✓; M✓ | H✓ | H✓ | H✓; R× | H× | 53, 61, 28, 60 | ||
| Immune | ||||||||
| Spleen | R✓ | R× | 53 | |||||
| Monocytes/macrophages | H✓; M× | H±; M× | H✓; R✓; M✓ | 47, 35, 3, 20, 86 | ||||
| Lymphocytes | H✓ | H✓ | H± | M✓ | 90, 66 | |||
| Thymus | R✓* | R✓* | 8 | |||||
| Bone | ||||||||
| Cartilage | R✓ | R:♂✓,♀× | R× | 63 | ||||
| Bone | H✓ | H✓ | H✓ | H± | R× | 80, 23 | ||
| Skin | ||||||||
| Genital skin | H✓ | H× | H✓ | H✓ | H✓; R✓ | 14, 57, 65, 77 | ||
| Non-genital skin | H✓ | H✓ | H± | H✓ | H✓; R✓ | 14, 76, 62, 36, 65, 16 | ||
| Hair follicle | H✓ | H✓ | H✓ | H✓ | H✓ | 16, 17, 68 | ||
| Sweat gland | H✓ | 67 | ||||||
| Gastrointestinal | ||||||||
| Stomach | R✓ | R× | 53 | |||||
| Intestine | R✓ | R✓ | H✓; R✓ | 52, 18, 53 | ||||
| Neurological | ||||||||
| Hypothalamus | H✓; R✓; M✓ | H✓; R✓ | R✓ | H×; R× | H× | R✓ | 30, 78, 56, 65, 31, 27 | |
| Pituitary | H×; R✓ | H×; R✓ | R✓ | H×; R× | H× | R✓ | 30, 65, 87, 54 | |
| Thalamus | M✓(G, g) | R✓; M✓(G, g) | R✓ | 56, 31, 2 | ||||
| Hippocampus | H✓; M×(G); M✓(g) | R✓; M×(G); M✓(g) | H× | R✓ | 44, 56, 31, 74, 2 | |||
| Cortex | H✓; R✓; M×(G); M✓(g) | H✓; R✓; M×(G); M✓(g) | H✓ | H×; R✓ | H× | R✓ | 44, 56, 31, 74, 73, 2, 79 | |
| Medulla oblongata | H✓ | H✓ | H✓ | H✓ | R✓ | 78, 31 | ||
| Pons | H✓ | H× | H× | H× | R✓ | 44, 78, 65, 31 | ||
| Amygdala | M(g)✓ | M(g)✓ | R✓ | 31, 2 | ||||
| Corpus callosum | M× | M× | R✓ | 44, 2 | ||||
| Striatum | M(G)✓ | M(G)✓ | 2 | |||||
| Cerebellum | H✓; M✓(G); M± (g) | H✓; M✓(G); M±(g) | H× | H× | R✓ | 44, 78, 65, 31, 2 | ||
| Spinal cord | R✓ | R✓ | R✓ | R✓ | R✓; M✓ | 21, 39, 58, 55 | ||
| PN/paraganglia | R✓ | R✓ | 45, 88 | |||||
| Sensory | ||||||||
| Olfactory bulb | R✓; M±(G); M✓(g) | R✓; M±(G); M✓(g) | R✓ | 31, 29, 2 | ||||
| Eyes | H✓ | H✓ | H✓ | 84, 64 | ||||
| Reproductive | ||||||||
| Prostate | H✓; R✓ | H×; R✓ | H✓; R✓; M± | H✓; R✓ | H✓; R✓ | R✓; M✓ | H✓ | 22, 53, 65, 41, 72 |
| Testes | H×; R✓ | H× | M✓ | H✓; R✓ | H✓ | M× | M✓ | 71, 65, 42 |
| Epididymis | H✓; R✓; M✓ | H×; R✓ | R✓; M✓ | H✓; R✓; M✓ | H✓; R✓ | R✓; M✓ | H✓ | 53, 78, 41, 11, 43, 81 |
| Vas deferens | R✓ | R✓ | H✓; R✓; M✓ | 15, 69, 26, 65 | ||||
| Seminal vesicles | H×; R✓ | H×; R✓ | M× | H✓; R✓ | H✓; R✓ | M× | 65, 41, 59 | |
| Ovary | H✓; R✓; M✓ | H× | H✓ | H✓; R×; M✓ | H× | H✓ | 4, 53, 78, 65, 49, 25, 37 | |
| Vagina | H✓; M✓ | H✓; M✓ | R✓ | 19, 7, 6, 37 | ||||
| Uterus | H✓; M✓ | H✓; M✓ | H✓; M± | H✓ | R✓ | 40, 7, 24, 37 | ||
| Placenta | M✓ | H✓; M✓ | H✓ | H✓ | H✓ | R✓ | 9, 46, 12, 40, 82 | |
| Breast | H✓ | H✓ | H✓ | H✓ | H✓; R✓; M✓ | 50, 34, 1, 85, 75 | ||
5αR1, 5α reductase type 1; 5αR2, 5α reductase type 2; H, human; R, rat; M, mouse; ✓, present; ×, absent; ±, very low levels; IC, either immunocytochemistry or immunohistochemistry; G, gamma aminobutyric acid; g, glutaminergic neurons; PN, peripheral nerves. Activitya=demonstration of substrate → product in conditions designed for optimal isozyme action. Activityb=ex vivo or in vivo demonstration of 5α reduction of substrate. *mRNA identified but isozyme not specified. 1Abulhajj & Klang (1982); 2Agis-Balboa et al. (2006); 3Araneo et al. (1991); 4Backstom et al. (1986); 5Barat et al. (2007); 6Berman et al. (2003); 7Blom et al. (2001); 8Borlak et al. (2004); 9Chan & Leathem (1975); 10Colby & Kitay (1972); 11Delarminat et al. (2011); 12Dombroski et al. (1997); 13Drake et al. (2005); 14Dube et al. (1975); 15Dupuy et al. (1979); 16Eicheler et al. (1995); 17Eicheler et al. (1998); 18Eiknes et al. (1983); 19George (1993); 20Hammer et al. (2005); 21Hauser et al. (1987); 22Houston et al. (1987); 23Issa et al. (2002); 24Ito et al. (2002); 25Jakimiuk et al. (1999); 26Jeanfaucher et al. (1986); 27Karolczak et al. (1998); 28Kimura et al. (2003); 29Kiyokage et al. (2005); 30Lephart (1993); 31Li et al. (1997); 32Livingstone et al. (2000); 33Livingstone et al. (2009); 34Lloyd (1979); 35Lofthus et al. (1984); 36Luu-The et al. (1994); 37Luu-The et al. (2005); 38MacKenzie et al. (2008); 39Maclusky et al. (1987); 40Mahendroo et al. (1997); 41Mahendroo et al. (2001); 42Mahendroo et al. (2004); 43Mahoney et al. (2011); 44Melcangi et al. (1988); 45Melcangi et al. (1990); 46Milewich et al. (1979); 47Milewich et al. (1983); 48Milewich et al. (1987); 49Milewich et al. (1995); 50Mori et al. (1978); 51Mowszowi & Bardin (1974); 52Nienstedt et al. (1980a,b); 53Normington & Russell (1992); 54Nowak (2002); 55Patte-Mensah et al. (2004); 56Pelletier et al. (1994); 57Pinsky et al. (1978); 58Pozzi et al. (2003); 59Pratls et al. (2003); 60Provost & Tremblay (2007); 61Provost et al. (2002); 62Randall et al. (1982); 63Raz et al. (2005); 64Rocha et al. (2000); 65Russell & Wilson (1994); 66Samy et al. (2001); 67Sato et al. (1998); 68Sawaya & Price (1997); 69Seethalakshmi et al. (1982); 70Seo et al. (2009); 71Sheffield & O'Shaughnessy (1988); 72Shirakawa et al. (2004); 73Steckelbroeck et al. (2001); 74Stoffel-Wagner et al. (2000); 75Suzuki et al. (2001); 76Takayasu et al. (1980); 77Thiele et al. (2005); 78Thigpen et al. (1993b); 79Torres & Ortega (2006); 80Turner et al. (1990); 81Viger & Robaire (2011); 82Vu et al. (2009); 83Wake et al. (2007); 84Weinstein et al. (1991); 85Wiebe et al. (2000); 86Yang et al. (2011); 87Yokoi et al. (1996); 88Yokoi et al. (1998a); 89Yokoi et al. (1998b); 90Zhou et al. (1998); 91Zyirek et al. (1987)
With such a wide distribution of 5α-reductase expression, it follows that 5α-reduced steroids can be synthesised in many tissues, including sites that are pharmaceutical targets of GCs, as well as sites of their adverse effects. This implies that not only are these natural occurring GCs important in health and disease but 5α reduction may yield metabolites exerting effects in many organ systems.
Substrates
Both 5αRs irreversibly catalyse the reduction of the steroidal Δ4–5 double bond:
5αRs have an absolute requirement for NADPH and are unable to utilise NADH as an alternative (Frederiksen & Wilson 1971). Insights into the NADPH-binding domain and also interactions between substrates and the active site have been gained through site-directed mutagenesis and also investigation of the characteristics of proteins translated in patients with natural mutations in 5αR2 (Wigley et al. 1994, Wang et al. 1999). 5α-Reduced dihydrosteroids are rapidly reduced at the 3 keto position by 3α or 3β-hydroxysteroid dehydrogenase, enzymes of the aldo-keto reductase 1C family. The ratio of 3α vs 3β metabolites varies greatly in a tissue-dependent manner but, in the liver, 3α-reduced products are formed predominantly (Steckelbroeck et al. 2004). In human and rat tissues, the pH optima allows biochemical differentiation between these two isozymes in vitro, with 5αR1 having a pH optimum of 6–8.5 and 5αR2 having a pH optimum around 5 (Andersson et al. 1991). Half-lives of both enzymes are in the order of 20–30 h (Russell & Wilson 1994), but this may be altered in disease (Wigley et al. 1994).
Both 5α-R isozymes accept a range of 3-oxo-4-ene steroids as substrates (Andersson & Russell 1990, Thigpen et al. 1993a), including many hormonally active steroids (Russell & Wilson 1994), including GCs, androgens, progestogens (Purdy et al. 1990) and mineralocorticoids. They also catalyse formation of allo-bile acids (Tomkins 1956, Schefer et al. 1966), which are present in the foetus and, in the adult, more prevalent in liver disease (Mendoza et al. 2003). Using rat liver enzyme, McGuire et al. (1960) demonstrated chemical substituents that prevented catalysis including steroids unsaturated at C1-2, with a methyl substitution at C2 or C6 or halogenation at the 9α position. The last two modifications are typical of synthetic GCs (McGuire et al. 1960), suggesting that endogenous but not most pharmaceutical steroids are substrates for the enzymes.
Androgens are better substrates for 5αR2 than 5αR1, with low Kms of <0.1 μM (Normington & Russell 1992). Testosterone is also the most researched substrate of 5αR1, but progesterone is its preferred substrate, with a lower Km (Andersson & Russell 1990, Normington & Russell 1992). GCs are poorer substrates for both human isozymes and thus unable to achieve high velocities of metabolism (Andersson & Russell 1990); in the case of human type 2, the apparent Km for cortisol was measured as 3.6 μM in epididymal microsomes (Fisher et al. 1978). In the case of the rat, the Km values for reduction of cortisol and corticosterone are 18 and 16 μM, respectively, for type 1 and 0.376 μM for type 2 (Normington & Russell 1992). The Km for 5α-reduction of cortisol is slightly lower than that for 5β-reduction measured in human hepatic cytosol (27 μM; Iyer et al. 1990), but these routes appear to make balanced contributions to metabolic clearance of steroids by first pass metabolism. 3α,5α-Reduced cortisol metabolites represent just under half of urinary tetrahydro-metabolites of cortisol synthesised in the liver (Fukushima et al. 1960), with 3α,5β-reduced metabolites comprising the rest. Similarly, metabolites of corticosterone of both isomeric configurations are detected in rodent urine (Livingstone et al. 2000, Shackleton et al. 2007).
Cortisone can also be metabolised by 5αRs (McGuire & Tomkins 1960, Gold & Crigler 1972) and, while it is a better substrate than cortisol (McGuire & Tomkins 1960), its 5α-reduced metabolites are not readily found in urine (Bush & Mahesh 1959). 5α-Tetrahydrocortisone, if present, is rapidly converted into 5α-tetrahydrocortisol (Bush & Mahesh 1959), possibly by 11β-hydroxysteroid dehydrogenase 1. This contrasts with the more skewed 5β-tetrahydrocortisone, which is not reduced at the 11 position and is consequently abundant in urine. Precursors for the synthesis of corticosteroid, 11-deoxycortisol and 11-deoxycorticosterone are also metabolised (Forchielli et al. 1955, McGuire & Tomkins 1960, Frederiksen & Wilson 1971) by 5αRs, indeed more rapidly than cortisol; McGuire & Tomkins (1960) found similar rates of metabolism of DOC as androstenedione using rat liver microsomes and this may help explain the suggestion that the activity of 5α-reductases in the adrenal gland plays part of a mechanism to regulate steroidogenesis (Carsia et al. 1984).
Actions of substrate vs product
Modification by 5α or 5β reduction results in distinct orientations of the A-ring, with the 5α confirmation adopting a planar ‘trans’ structure, whereas the 5β product assumes a skewed ‘cis’ orientation. The Δ4–5 double bond in the parent is more planar than the four to five single bonds in the 5α-reduced metabolite (Askew et al. 2007). These structural differences influence the ability of metabolites to interact with the cognate receptor (Askew et al. 2007) and metabolising enzymes (Bush & Mahesh 1959) and thereby critically influence their ability to trigger downstream signalling pathways. It has long been recognised that 5α-reduced androgens are more potent than the parent steroid, binding to the AR with greater affinity, and also dissociating from it three times more slowly (Wilson & French 1976, Chang et al. 1988, Lubahn et al. 1988, Askew et al. 2007). Likewise, 5α-dihydroprogesterone is a ligand for the progesterone receptor (Pasqualini & Nguyen 1980) and is the principle progestogen in some species (Meyer et al. 1997a). Its subsequent 3α-reduced metabolite, allopregnenolone (Uzunova et al. 2006), promotes inhibitory neuronal tone via an independent mechanism mediated by the GABA-A receptor (Puia et al. 1990). 5α-Reduced metabolites of aldosterone have weak mineralocorticoid activities (Sekihara et al. 1978, Kenyon et al. 1983, 1985, Gorsline et al. 1986, Morris 1986). In contrast to other steroids, metabolism of GCs by 5αR has generally been thought to inactivate these steroids prior to excretion and, throughout the years, there have only been sporadic reports of 5α-reduced GCs exerting effects through GR.
A few studies have suggested that the 5α-reduced metabolites of cortisol or corticosterone can bind GR, first demonstrated by Baxter & Tomkins (1971), who showed that 5α-dihydro-cortisol was able to displace tritiated dexamethasone from cytosolic GR, but more weakly than other endogenous steroids. In similar experiments, even weaker displacement of tritiated dexamethasone by 5α-dihydrocorticosterone (5αDHB) was demonstrated by Carlstedt-Duke et al. (1977). While McInnes et al. (2004) concurred that 5α-DHB was a weak GR ligand, they also demonstrated that 5α-tetrahydrocorticosterone (5αTHB) could displace dexamethasone with a similar Kd as corticosterone (Fig. 3A). Recently, 5α-DHB has also been suggested as a ligand for a putative, alternative DHB receptor (Sheppard et al. 1998).
5α-Reduced glucocorticoid binds and selectively activates GR. (A) 5α-Tetrahydrocorticosterone (5αTHB) and 5α-dihydrocorticosterone (5αDHB) (overnight incubation with concentration range from 10−5 to 10−9 M) displaced tritiated dexamethasone (100 nM) from glucocorticoid receptors in rat hepatic cytosol. The Kds for binding of 5αTHB (268 nM) and 5αDHB (336 nM) were similar to that of corticosterone (153 nM) and higher than dexamethasone (38 nM; n=6/treatment). (B) 5αTHB, but not 5αDHB, induced transcription of tyrosine aminotransferase (TAT) to a lesser extent than corticosterone (B) when incubated for 24 h in H4iiE cells (rat hepatoma cells). Abundance of transcript was quantified by northern blot and normalised for that of U1, n=6/treatment. *P<0.05 vs vehicle (veh), #P<0.05 vs B. Following chronic infusion of steroids (50 μg/day, 2 weeks) to mice (n=10–12/group): (C) corticosterone, but not 5αTHB, impaired glucose tolerance, as demonstrated by increased insulin concentrations (P<0.001 vs vehicle) after a glucose tolerance test (2 g/kg body weight i.p.) performed in mice following a 6 h fast. Insulin was quantified by immunoassay. (D) 5αTHB caused immune suppression to a similar extent to corticosterone, assessed by a significant reduction (P<0.01) in the ability of lipopolysaccharide (LPS; 0.01–100 ng/ml)) to induce release of interleukin 6 (IL6) following incubation (24 h) from cells in whole blood harvested at cull. IL6 was quantified by immunoassay. Data are mean±s.e.m. Data presented in A and B are adapted from those originally published in the McInnes KJ, Kenyon CJ, Chapman KE, Livingstone DEW, Macdonald LJ, Walker BR & Andrew R 2004 5α-Reduced glucocorticoid metabolites, novel endogenous activators of glucocorticoid receptors (GR). Journal of Biological Chemistry 279 22908–22912 © the American Society for Biochemistry and Molecular Biology. Data presented in C and D were originally published in the Yang CA, Nixon M, Kenyon CJ, Livingstone DEW, Duffin R, Rossi AG, Walker BR & Andrew R 2011 5α-Reduced glucocorticoids exhibit dissociated anti-inflammatory and metabolic effects. British Journal of Pharmacology. (In press) (doi:10.1111/j.1476-5381.2011.01465.x).
Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0318
The potency and efficacy of 5α-reduced corticosterone metabolites have been compared to the parent steroid in vitro, exploring functional endpoints that depend on different modalities of GR receptor signalling. Signalling by transactivation through conventional GR dimerisation can be modelled through activation of the MMTV promoter linked to a luciferase reporter (Grange et al. 2001). This assay is frequently used as a screening tool for metabolic side effects during development of dissociated GR ligands (Schake et al. 2004). 5αTHB is a weaker activator of this promoter than the parent steroid and stimulates transcription of the TAT gene in hepatocytes to a lesser extent than the parent steroid (McInnes et al. 2004; Fig. 3B). Similar early reports by Samuels & Tomkins (1970) classified 5α-dihydrocortisol as a ‘sub-optimal inducer’ (i.e. a partial agonist) of TAT activity in hepatoma cells. These findings were supported by limited responses of other hepatic genes transactivated by GC (Danesch et al. 1987, Ruppert et al. 1990); 5αTHB induces PEPCK weakly (McInnes et al. 2004), 5α-dihydrocortisol lacks efficacy to stimulate activity of tryptophan oxidase (Carlstedt-Duke et al. 1977) and achieved sub-optimal induction of hepatocyte adhesion (Ballard & Tomkins 1969). Overall, the weak effects of these metabolites caused interest in their potential biological role to wane.
These small inductions of gene transcription were corroborated by lack of metabolic side effects when 5αTHB was administered in vivo to mice, e.g. in TAT activity or expression unlike those observed when the endogenous active hormone, corticosterone was administered (Yang et al. 2011). The pathophysiological correlation of these changes is impaired insulin sensitivity, as demonstrated by increased plasma insulin levels during glucose tolerance testing, in the corticosterone-treated animals, which did not develop when 5αTHB was administered instead (Fig. 4A). This lack of metabolic disruption has also been demonstrated in rats, where administration of 5α-reduced dihydroglucocorticoids (5α-dihydrocortisol and 5α-DHB) for a week actually lowered plasma insulin and hepatic PEPCK activity (Golf et al. 1984). These latter effects may arise from suppression of synthesis of corticosterone, since, in both rats (McInnes et al. 2004) and mice (Yang et al. 2011), 5αTHB suppresses the HPA axis. Ex vivo, 5α-DHB impairs the development of long-term potential in neurones of the dentate gyrus (Dubrovsky et al. 1987). In contrast to their limited metabolic actions, a recent study has shown that 5α-reduced steroids possess anti-inflammatory abilities, in vitro and in vivo: 5αTHB suppressed release of pro-inflammatory cytokines in cultured bone marrow-derived macrophages, induced immune suppression when administered chronically, and also alleviated the induction of inflammation in response to challenge (Yang et al. 2011). This combination of activities suggests that 5α-reduced GCs have a different spectrum of biological actions from their parent steroids and poses the question of why changing the chemical nature in the steroidal A-ring might cause this change in profile.
Binding of testosterone and 5α-dihydrotestosterone (5αDHT) in the androgen receptor ligand-binding domain (AR-LBD). When bound with AR, testosterone (A) and 5αDHT (B) are positioned near the side chain of Arg-752 (R752), a helix 5 residue required for ligand binding. The bond between the 3-keto-O on the steroid and R752 is influenced by the 4–5 double bond in testosterone, which imparts a more planar structure to testosterone than that seen in 5αDHT (C, dashed arrow), allowing more favourable H-bonding. The presence of structural water HOH1 permits a H-bond network between the steroid and key residues in the AF2 transactivation domain of the receptor. In particular, a bridged H-bond with Met-745 (M745), a residue that lies directly above the A-ring of the steroid, is formed. The structure of this bond is believed to be important as M745 projects towards Leu712 (L712), a proximal residue in the AF2 domain crucial in mediating contacts with residues within accessory molecules. Alterations in the structural integrity of L712 as a result of this projection (C, Block arrow) are believed to directly affect the binding of key cofactors essential for gene transcription. 5αDHT has been suggested to impart greater structural integrity to L712, stabilising cofactor binding and thus enhancing AR activity (Askew et al. 2007). Images were generated with ICM-Pro Software (MolSoft, San Diego, CA, USA) for testosterone with AR-LBD (Protein Data Bank Code 2Q7I) and 5αDHT with AR-LBD (Protein Data Bank Code 1T63).
Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0318
Differential interactions between ligand and receptor can influence the spectrum of down-stream signalling and the ability of the occupied receptor to recruit co-activators and co-repressors. Dexamethasone (Bledsoe et al. 2002), but not corticosterone or its 5α-reduced metabolites, has been crystallised with human GR. Nonetheless, insights into potential, discriminant interactions of parent and metabolite can be inferred through parallels with 5α-reduced androgens, which have been studied bound with the recently crystalised AR (Askew et al. 2007). GR and AR belong to the same superfamily of nuclear receptors, sharing many structural and functional features (Bledsoe et al. 2002). The ligand-binding domain (LBD) contains a binding pocket specific for the cognate ligand, as well as the second activation function (AF2; Bledsoe et al. 2002, McMaster & Ray 2007). On ligand binding, interactions of AF2 with a broad range of motifs are permitted (Hur et al. 2004), including the p160 family of co-activators (Voegel et al. 1996, Heery et al. 1997, Ma et al. 1999, He et al. 2006), and differences have been proposed in the profile interactions with AF2 domain following binding of testosterone or 5αDHT with AR.
Testosterone is a less potent androgen than 5αDHT, with a tenfold higher concentration needed to achieve equipotent AR-mediated effects. Structural data suggest that the reduced number of protons present in testosterone compared with 5αDHT ensures a superior hydrophilic profile and greater H-bonding potential within the LBD of AR (Askew et al. 2007; Fig. 4). The altered conformation, with a more planar Δ4–5 double bond present in testosterone relative to 5αDHT, allows testosterone to accept a bridged H-bond with Met745, a residue in the AF2 domain of the AR that lies directly above the steroidal A-ring. In this setting, greater H-bonding is detrimental to agonist activity, with interactions weakened or absent in the presence of 5αDHT. The projection of this bridged H-bond is towards Leu712, a proximal residue in the AF2 domain crucial in mediating contacts with residues within accessory molecules (Ghali et al. 2003). 5αDHT has been suggested to impart greater structural integrity to Leu712, stabilising cofactor binding and thus enhancing AR activity (Askew et al. 2007).
Due to the homology between AR and GR, similar differential interactions may be proposed for 5α-reduced GCs and their parent steroid, potentially causing different subsets of co-activators and repressors to be recruited. Within the AF2 domain in GR, Arg611, Met605 and Gln711 are homologous to Arg752, Met745 and Gln711 in AR. However, in GR, a different hydrophobic amino acid, valine, is present in GR in place of Leu712 in AR, restricting flexibility of interactions. The abilities of ligands to induce different conformations in GR and recruit different proteins (e.g. TIF2 or NCoR) have been investigated for the agonist, dexamethasone and the antagonist, mifepristone (RU38486; Schoch et al. 2010). Taken together, these reports support the concept that the exact topography of the interactions of 5α-reduced steroids within GR-LBD may influence their profile of activity when bound with the receptor and may, in the future, provide an explanation for why 5α-reduced GCs retain some, but not all, actions of their parent GCs. Crystallisation of the metabolites with GR is required to add credence to this hypothesis and allow subsequent design of dissociated steroids using their structure and interactions as a template.
Roles in health and disease
While 5α-reduced GCs present a potential prototype for drug design, a question remains about their role in vivo. In the many tissues where 5α-reductases are expressed, this enzyme will regulate the proportions of the parent steroid and the metabolite, potentially modulating the balance of metabolic and anti-inflammatory actions. Given the high expression of the isozymes in liver and rapid inactivation of 5α-reduced GC by hepatic conjugation, circulating concentrations will be low (Furuta et al. 1998), predicting paracrine rather than endocrine signalling. Therefore, understanding the factors regulating the balance between parent and metabolite is crucial.
The ratio of urinary 5α- and 5β-reduced GC metabolites, primarily influenced by the liver, is altered in metabolic disease (Andrew et al. 1998, Fraser et al. 1999, Rask et al. 2001, 2002). Indeed, this was the fact that drew attention to the potential importance of these metabolic routes to regulate GC action. In obesity, the total daily production of GCs and the proportion of cortisol metabolised by 5α-reduction are increased (Andrew et al. 1998, Fraser et al. 1999, Rask et al. 2001, 2002); this can be corrected by weight loss (Johnstone et al. 2004, Tomlinson et al. 2008) and recapitulated in rats, where the liver can be identified as the major site of up-regulation (Livingstone et al. 2000). Again in rodents, insulin sensitisation ameliorates these changes (Livingstone et al. 2005). Interestingly, in humans with fatty liver, the proportion of cortisol metabolised by 5α-reduction is less (Westerbacka et al. 2003) and mice lacking 5α-reductase 1 develop fatty liver (Livingstone et al. 2008). These data suggest a pattern of hepatic metabolic dysfunction and susceptibility to inflammation under conditions of low activity of hepatic 5α-reduction of GCs.
Genetic variants in 5αRs have not been described in polygenic obesity or type 2 diabetes, and in particular, regulation of 5αR1 is poorly understood with little known about functional regulatory promoter elements. The most likely candidate for dysregulation in metabolic disease is insulin, although sex steroids and GCs also have powerful influences (Miller & Colas 1982, Horton et al. 1993, El-Awady et al. 2004). In hepatocytes, albeit under culture, insulin up-regulates the enzyme 5αR1, whereas 5αR2 is amplified primarily by feed-forward action of the metabolite (George et al. 1991).
Changes in GC clearance rates subsequent to up-regulation of hepatic metabolism cause adaptation in the set point of the HPA axis, which is fine-tuned to maintain circulating GC levels within a tight window. Increased GC production can be inferred from the adrenal hypertrophy evident in obese rodents, which is also ameliorated by insulin sensitisation. A similar association can be extrapolated in PCOS, where 5α-reduction of GCs is up-regulated (Stewart et al. 1990, Fassnacht et al. 2003, Tsilchorozidou et al. 2003) and insulin sensitisation normalises adrenal hyperactivity (Dunaif et al. 1996).
Conclusion
5α-Reduced GCs have received little attention in terms of their biological activity, despite other 5α-reduced steroids being well recognised as active hormones. They are formed in many tissues where GR is expressed and revisiting recent and previous literature allows one to propose a distinct profile of actions in comparison to the parent GC. They appear weak regulators of metabolism but retain other biological actions of pharmaceutical interest. To date, the spectrum of actions of 5α-reduced GC has not been investigated in bone.
A further avenue of interest in 5α-reduced GCs relates to the common pharmacological inhibition of 5αRs in clinical practice. 5α-Reductase inhibitors are used to treat benign prostate hyperplasia, a disease afflicting over half of elderly men. Although unlicensed, these drugs are also used to treat women with PCOS. It is possible that these patients will develop an imbalance in parent and 5α-reduced metabolites, leaving them susceptible to adverse metabolic effects and inflammatory changes, effects more marked with dutasteride, a new dual 5αR inhibitor. To date, there have not been any comprehensive metabolic studies in patients on these drugs (Amory et al. 2007).
Greater understanding of the role of 5α-reduced GCs in health, disease and therapeutics will provide important insights into the effects in patients on 5α-reductase inhibitors, as well as providing an exciting focus of research in the quest for novel selective GR modulators.
Declaration of interest
R A is an inventor of a relevant patent held by the University of Edinburgh.
Funding
This work was supported by the Wellcome Trust (grant numbers 072217/Z/03/Z, GR060707FR), British Heart Foundation (grant number RG/05/008), Chief Scientist Office (grant number CZB/4/642), Diabetes UK (grant number RD 05/0003028), Society for Endocrinology and Medical Research Council for Funding. R U is a recipient of the Graham Aitken Nuffield Postgraduate Travelling Scholarship.
Author contribution statement
M N, R U and R A co-wrote the manuscript, and R A collated the material.
Acknowledgements
We are grateful to Diego Cobice and Gregorio Naredo for their help with chemical figures.
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