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Vishal Bhoir, Marta Roldan Lazaro, Cristina Benito, Alexsandra Tifane Santos do Nascimento, Student

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 ===N-terminal Domain (NTD)(residues 1-555)===
 This region is required for full transcriptional activity <ref>PMID: 24909511 </ref>, because of its necessary presence for LBD activation <ref>PMID: 33076388</ref>. It is the most variable domain, the sequence and lengths of the polyglutamine (CAG) and polyglycine (GGC) repeats are highly variable in the human population. It has been shown that the length of the polyglutamine repeat region affects the folding and structure of this domain, shorter repeats generally impose a higher AR transactivation activity, whereas longer repeats cause reduced activity  <ref>PMID: 24909511 </ref>. In healthy people, one region of the AR gene shows up to 36 repeats of the CAG sequence. Patients with abnormally high numbers of CAG repeats can develop spinal muscular atrophy.
-===DNA-binding Domain (DBD) (residues 555-623)===
+===DNA-Binding Domain (DBD) (residues 555-623)===
 DBD is a cysteine-rich region that is the most highly conserved one of the steroid hormone nuclear receptor family <ref>PMID: 24909511 </ref>, but it has been shown that binding of selective androgen response elements (AREs) allow the specific activation functions of the AR. They facilitate direct DNA binding of the AR to the promoter and enhancer regions of AR-regulated genes, thereby allowing the activation functions of the N-terminal and ligand binding domains to stimulate or repress the transcription of these genes <ref>PMID: 27057074</ref>.
 AR is a dimer, like other steroid receptors, that binds to promoter DNA response elements consisting of two equal, common hexameric half-sites, separated by a 3 base-pair spacer <ref>PMID: 24909511 </ref>'''IMAGEN DEL DÍMERO''', and this domain is critical for AR function, because it plays a role in dimerization and binding of dimerized AR to select motifs on target DNA <ref>PMID: 33076388</ref>.
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 The DBD is linked to the ligand binding domain by a flexible hinge region (residues 623-665), which is a linker poorly conserved. Once in the nucleus, this region also interacts with the DBD to identify specific sequences for AR binding. It controls the AR activation and degradation. Consequently, mutations in the hinge region can lead to enhanced AR potency <ref>PMID: 33076388</ref>.
+===Ligand-Binding Domain (LBD) (residues 665-919)===
+The LBD, located at the C-terminal, is the main target of AR inhibitors <ref>PMID: 33076388</ref>. It consists of eleven α-helixes in the ligand binding pocket, with reposition upon androgen binding, converting into the activation function 2 (AF-2) domain. Unlike other nuclear receptors, the AR does not have H2, which is instead replaced by a long flexible linker <ref>PMID: 24909511 </ref>. The LBD binds motifs in the NTD and in AR-specific cofactors and coactivators. Moreover, LBD-LBD homodimerization of AR is essential in the proper functioning of the receptos <ref>PMID: 33076388</ref>.
+This domain has been structurally well characterized by crystallography and a number of mutations have been identified. It is important because not all mutations affect ligand binding, but some of them may disrupt androgen induced interaction of the N-terminal motif and C-terminal AF-2 <ref>PMID: 33076388</ref>.
+==Transcriptional Activation Function==
+Two transcriptional activation functions have been identified:
+-The ligand-independent AF-1 (residues 142-485): located in the NTD is constitutively active. It is the main region responsible for mediating AR transcription. This region contains two separable transcription activation units that are indispensable for full activity of the AR <ref>PMID: 24909511 </ref>.
+-The ligand-dependent AF-2: is located in the ligand binding domain <ref>PMID: 27057074</ref>. H12 forms the core of this region and acts as a lid to close the LBP upon agonist binding <ref>PMID: 24909511 </ref>. It is important for forming the coregulator bindings site as well as mediating direct interactions between the N-terminal and ligand binding domains. Key differences in the contribution of specific conserved residues in the AF-2 core domain between the AR and other steroid hormone nuclear receptors have been identified, it would explain the differences in the structure and the function, as well as the coregulatory proteins they interact with <ref>PMID: 27057074</ref>.
+=Mechanism of Action=
+==AR signaling==
+AR has two mechanisms of action: the DNA binding-dependent (genomic AR signaling) and the DNA binding independent (non-genomic AR signaling).
+'''[[Image:Example.jpg]]'''
+===DNA-Binding dependent actions of the AR===
+In the absence of ligand, the AR is in the cytoplasm and associated with heat-shock and other chaperone proteins. Testosterone is converted into DHT by 5α-reductase, with higher affinity to bind AR. When DHT binds AR, it displaces heat shock proteins, drives the interaction between the N and C terminal, and binds importin-α to translocate the ligand/AR complex into the nucleus.
+In the nucleus, the receptor dimerizes and binds to AREs in the promoter regions of target genes. At the promoter, the AR is able to recruit members of the basal transcription machinery in addition to other coregulators to facilitate transcription <ref>PMID: 24909511 </ref>. AR activity is not only regulated by ligand binding and DNA binding but also by intramolecular interactions between functional domains, by homodimerization and by interactions with cofactors (4).
+This leads to the initiation of transcription, cell proliferation and survival, and negative feedback to inactivate AR transcription <ref>PMID: 27057074</ref>.
+'''[[Image:Example.jpg]]'''
+===Non-DNA Binding dependent actions of the AR===
+It has been shown that the androgen/AR complex activates 2nd messenger pathways including ERK, Akt and MAPK and that it interferes with several key proteins including forkhead box protein A1 (FOXA1), PI3K and receptor tyrosine kinases, including ERBB2 and ERBB3. These effects occur within seconds to minutes of androgen treatment <ref>PMID: 27057074</ref><ref>PMID: 28301631</ref>.
+There are studies that suggest that some of the non-DNA binding-dependent actions of androgens are mediated via the activation of membrane-bound protein receptors. For instance, the iron-regulated transporter-like protein 9 (ZIP9) mediates the androgen-induced apoptosis of ovarian follicle cells, prostate and breast cancer cells <ref>PMID: 27057074</ref>.
+Although the physiological significance of the non-DNA binding-dependent actions of the AR is not yet fully defined, it has been proposed that they may oppose the DNA binding-dependent actions and serve as a brake to fine-tune androgen action in target tissues <ref>PMID: 27057074</ref>.
+===Ligand-Independent actions of the AR===
+It has been demonstrated that AR has the potential to be activated through ligand-independent mechanisms by a number of different growth factors, via phosphorylation of the AR or following interaction with co-activators <ref>PMID: 27057074</ref>.
+=Function=
+AR is expressed in many tissues, so androgens have been documented to have significant biological actions in bone, muscle, prostate, ovaries, endometrium, bladder, skin, cardiovascular, immune, neural and hematopoietic systems <ref>PMID: 27057074</ref><ref>PMID: 29481861</ref>.
+Androgens have a role in behavior and cognition in neuronal cells in the CNS (III). It has also been shown they regulate hair growth, sebum production and secretion, wound healing and cutaneous barrier formation in the skin <ref>PMID: 28912032</ref>.
+The absence of AR has an impact on the fertility in granulosa cells in the ovary and it also affects the myometrial cell growth in uterine glandular epithelial cells in the endometrium <ref>PMID: 29481861</ref>.
+==Diseases==
+Drugs targeting the AR has the potential to be used in prostate cancer, benign prostatic hyperplasia (BPH), osteoporosis, breast cancer, hypogonadism, conditions where cachexia is a consequence of the disease state (HIV, cancer, immobilization), urinary incontinence, muscle wasting conditions as Duchenne muscular dystrophy (DMD) and Alzheimer’s disease <ref>PMID: 30503797</ref><ref>PMID: 23457206</ref><ref>PMID: 32257854</ref><ref>PMID: 28624515</ref>.
+====Prostate Cancer====
+Androgen receptor is fundamental for the correct function, development of  the prostate <ref>PMID: 12237244</ref> having a critical role in the control of homeostasis between prostate cells differentiation and proliferation <ref>PMID: 24639562</ref>. It's widely accepted that androgen receptor plays an important role in prostate cancer cells due to the alteration of that equilibrium, shifting the AR to a more proliferative transcriptional program <ref>PMID: 24639562</ref>.
+====Alzheimer’s Disease====
+Androgen depletion is considered a significant risk factor for Alzheimer’s disease and circulating testosterone levels are inversely correlated with levels of ß-amyloid (ßA) in the brains of aged men <ref>PMID: 30503797</ref>.
+=Pharmacology=
+==Steroid==
+===Natural ligand: Testosterone===
+Aging and other factors are associated with a reduction of testosterone levels which could lead to late onset hypogonadism <ref>PMID: 32737921</ref>. The decrease of the testosterone levels and therefore its active metabolite levels (DHT) are related with several symptoms such as low libido, erectile dysfunction, skeletal muscular loss <ref>PMID: 23457206</ref><ref>PMID: 32737921</ref>, increased cardiovascular risk <ref>PMID: 32737921</ref>… To  treat those symptoms is used Testosterone Restitution Therapy (TRT) which has been associated with the improve in sexual function, increase in muscle mass and bone mineral density <ref>PMID: 34888506</ref>.
+One of the problems associated with the use of T as a therapeutic agent in TRT are the delivery method, tending to have low efficacy orally administered <ref>PMID: 23457206</ref><ref>PMID: 32257854</ref> and having some inconvenients with intramuscular injections or implants <ref>PMID: 23457206</ref>. Also, the use of this hormone as a treatment could triggered a lot of AR widespread around the body and a long-term exposure to a high dose could lead to related side effects like erythrocytosis <ref>PMID: 23457206</ref><ref>PMID: 32257854</ref>, dyslipidemia, hepatotoxicity <ref>PMID: 32257854</ref> and in some clinical trials it has been described an increase in cardiovascular risk <ref>PMID: 34888506</ref><ref>PMID: 32737921</ref>.
+Due to all these problems, some institutions like the FDA warn about the safety issues related with this therapy assessing the reduction of its use <ref>PMID: 34888506</ref><ref>PMID: 32737921</ref>. However, other agencies like the EMA supported by the European Academy of Andrology establish the practical use of this therapy in men’s hypogonadism <ref>PMID: 34888506</ref><ref>PMID: 32737921</ref>. So, there is still some controversy about its use, and it’s still currently studied in clinical trials<ref>PMID: 34888506</ref>.
+===Antagonist: Steroid ARA===
+The development of these drugs were one of the first approaches to treat prostate cancer, targeting AR activity by having a structure with an steroidal skeleton <ref>PMID: 24639562</ref>.
+This kind of antiandrogens have another steroid receptor affinity (glucocorticoids receptor, progesterone receptor…) having low efficiency and some side effects like hepatotoxicity and increased cardiovascular risks <ref>PMID: 24639562</ref>. Some examples are cyproterone acetate (CPA) <ref>PMID: 24639562</ref><ref>PMID: 15833816</ref><ref>PMID: 16841196</ref> or megestrol acetate <ref>PMID: 24639562</ref>.
+===Agonist: Anabolic Androgen Steroids (AAs)===
+These drugs have been produced since the middle of the 20th century <ref>PMID: 33148520</ref>. They have anabolic activity which improves muscular mass and physical function. However, their uncontrolled use and abuse lead to several side effects like: testicular atrophy, alopecia, gynecomastia in the case of males, and clitoral hypertrophy, menstrual irregularities in the case of women. Men and women can experience mood disorders and the chronic abuse could result in high risk of suffering cardiovascular disease and prostate cancer <ref>PMID: 33148520</ref>.
+Because of the doping scandals in the athlete community, the World Anti-Doping Agency (WADA) has prohibited them <ref>PMID: 33148520</ref>. This creates the need to discover androgens that have beneficial anabolic activity with reduced androgenic activity.
+==Non-Steroid==
+===Selective Androgen Receptor Modulators (SARMs)===
+Steroid androgens can be associated with a high rate of adverse effects, which limits their widespread clinical use. To overcome these side effects, SARMs were developed.
+SARMs are small molecule drugs that manipulate the AR function in different tissues <ref>PMID: 30503797</ref>. They can act as both agonist and antagonist, making them potential to treat AR-related diseases.
+These non-steroidal drugs normally can be administered orally or using a transdermal injection <ref>PMID: 23457206</ref><ref>PMID: 32257854</ref> having better compliance and there are not affected by 5α-reductase (limiting its androgenic risk effects) and aromatase (limiting its estrogenic risk effects) <ref>PMID: 23457206</ref><ref>PMID: 32257854</ref>. Those characteristics help to the reduction of side effects related with the use of natural androgens and the tissue selectivity of these drugs make them a suitable option to treat a great group of diseases skipping the risk related with the use of TRT <ref>PMID: 23457206</ref><ref>PMID: 32257854</ref><ref>PMID: 28624515</ref>.
+Also, some SARMs could be a suitable option to achieve the improvement in anabolic activity and muscular density obtained by the use of AAs without the unwanted side effects associated with their androgenic action of those drugs <ref>PMID: 33148520</ref>.
+====Mechanism of SARMs====
+Currently SARMs tissue selectivity is still under research <ref>PMID: 28624515</ref><ref>PMID: 33148520</ref>. There is no consensus on SARMs mechanisms of action. However, there are two hypotheses that could explain it:
+It could be related with their non-steroidal composition and with the fact that they are unaffected by 5α-reductase <ref>PMID: 32257854</ref><ref>PMID: 28624515</ref><ref>PMID: 33148520</ref> which promotes the interaction of AR with tissue-specific coactivators.
+The way SARMs bind to the AR is what primarily enhances or represses their effect. Each SARM-AR complex has a different conformation and tissues have unique patterns of AR expression, co-regulatory proteins levels and transcriptional regulation <ref>PMID: 30503797</ref>.
+When a ligand promotes interactions between the N- and C-terminal AR domains, the AR is maximally active. The ability to reduce N/C interactions is the hallmark of SARMs that display antagonisms in androgenic tissues <ref>PMID: 30503797</ref>.
+====Diseases that could be treated with SARMs====
+SARMs may one day play a role in the treatment of cognitive disorders, such as '''Alzheimer's disease'''. Androgens facilitate the reduction of deleterious ß-amyloid (ßA) plaques, upregulating the expression of ßA-degrading neprilysin and they promote synapse formation and neurogenesis, upregulating brain derived neurotrophic factor <ref>PMID: 33148520</ref>.
+AR in '''breast cancer''' likely confers survival advantage by modulating ER signaling, which may reduce the risk of metastasis and aggressive disease. There is a clinical trial seeking to evaluate pembrolizumab and enobosarm co-therapy for the treatment of AR positive metastatic triple negative breast cancer <ref>PMID: 30503797</ref>.
+'''Urinary incontinence''' denotes involuntary bladder urine leakage amongst women commonly with decreased pelvic muscle strength. As the pelvic floor muscles contain high levels of AR, it is a relevant target for SARM therapy <ref>PMID: 33148520</ref>.
+====Side effects====
+Despite the consistent effect demonstrated by SARMs on lean body mass accrual, reductions in high-density lipoprotein (HDL) with even low doses seem to be an important concern with these compounds, though it occurs to a lesser extent compared to testosterone <ref>PMID: 32476495</ref>.
+SARMs administration has also been related to hepatotoxicity and some compounds have shown liver enzymes alterations, the most common adverse events being increases in alanine transaminase and aspartate transaminase <ref>PMID: 32476495</ref>.
+The anabolic effects of SARMs and their lack of androgenic side effects have made them of great interest to the bodybuilding community and create the potential for abuse among competitive athletes <ref>PMID: 30503797</ref>.
+===Antagonist===
+These kinds of drugs were developed with the objective to avoid the secondary effects associated with cross reactivity of steroidal ARA, increasing the selectivity and the affinity to the androgen receptor, limiting the association with other steroids nuclear receptors <ref>PMID: 15833816</ref>. Also, their non-steroidal structure improved oral bioavailability being another advantage in comparison with steroidal ARA <ref>PMID: 15833816</ref>. Some examples are flutamide, bicalutamide <ref>PMID: 24639562</ref><ref>PMID: 15833816</ref><ref>PMID: 12015321</ref><ref>PMID: 21506597</ref><ref>PMID: 16841196</ref><ref>PMID: 30209899</ref> or apalutamide (ARN-509) <ref>PMID: 24639562</ref><ref>PMID: 30209899</ref>.
+====Bicalutamide====
+R-Bicalutamide, marketed as Casodex <ref>PMID: 24639562</ref><ref>PMID: 16841196</ref>, is one of the most stable and tolerated androgen receptor antagonists used in the treatment of prostate cancer <ref>PMID: 24639562</ref><ref>PMID: 15833816</ref><ref>PMID: 28971898</ref>, belonging to the first generation of antiandrogens developed <ref>PMID: 24639562</ref><ref>PMID: 35245614</ref>. It is a competitive antagonist <ref>PMID: 12015321</ref><ref>PMID: 35245614</ref><ref>PMID: 28971898</ref> which binds to the LBD producing a transcriptionally inactive androgen receptor <ref>PMID: 12015321</ref>. However, it seems that the long-term use of these drugs and other first generation antiandrogens leads to withdrawal syndrome in prostate cancer resistant to castration patients <ref>PMID: 24639562</ref><ref>PMID: 16841196</ref>. In many cases associated androgen receptor mutations like W741L that can switch the mechanism of action of the drug from antagonist to agonist or partial agonist <ref>PMID: 24639562</ref><ref>PMID: 15833816</ref><ref>PMID: 35245614</ref><ref>PMID: 21506597</ref><ref>PMID: 16841196</ref>.
+Although bicalutamide has been patented since 1982 and approved to be clinical used by the FDA since 1995 <ref>PMID: 15833816</ref>, its mechanism of action it's still a debate, because the X-ray structure of the wild-type androgen receptor binded to an antagonist is not yet solved <ref>PMID: 35245614</ref>.
+Changes in the conformation of the androgen receptor due to association with antagonists have been hypothesized to be similar to those produced in the steroid receptor family <ref>PMID: 24639562</ref><ref>PMID: 35245614</ref>.
+When an agonist or a ligand binds to the LBD it seems that it induces a conformation of the steroid receptor which makes H12 close off the pocket of LBD allowing the union of cofactors so, at the end, permitting the steroid receptor function allowing the DNA transcription <ref>PMID: 24639562</ref>. Although, when an antagonist is binded, H12 seems to be more separated to the LBD, disabling the binding of coactivators <ref>PMID: 24639562</ref> and the migration of the nuclear receptor into the nucleus <ref>PMID: 35245614</ref>.
+Nonetheless, the AR has some structural singularities that may not let this change of conformation, being the most important the additional C-terminal region in H12 anchored to the receptor by the formation of a ß-sheet, limiting its movement <ref>PMID: 24639562</ref><ref>PMID: 35245614</ref>. Due to this structural difference, in silico approaches have suggested that the antiandrogen effect of bicalutamide may be produced by the instability of the homodimer <ref>PMID: 35245614</ref>. That may lend to the homodimer dissociation preventing the transcriptional activity of the AR explaining the mechanism of action of this drug <ref>PMID: 35245614</ref>. Also, in silico analysis have shown that the W741L mutation leads to a bicalutamide-AR homodimer more stable, which may make some insight into the withdrawal syndrome observed in bicalutamide treatment <ref>PMID: 35245614</ref>.
+It is mandatory to understand by future research the whole mechanism of action of the current antiandrogens clinically used, with the objective of developing new drugs which can escape to the antagonist-agonist switch seen by bicalutamide, or other antiandrogens like flutamide.
+One example of this is apalutamide, a non-steroidal second generation antiandrogen <ref>PMID: 24639562</ref><ref>PMID: 30209899</ref> approved for use in non metastatic castration resistant prostate cancer patients by the FDA in 2018 <ref>PMID: 30209899</ref>. See also the SPARTAN study <ref>PMID: 29420164</ref>: [https://clinicaltrials.gov/ct2/show/NCT01946204]. This new drug has promising uses but it is still associated with side effects like an increased level of falls in patients with the treatment vs placebo <ref>PMID: 36209239</ref>.