WO2006081658A1 - In silico screening for shbg binding ligands via pharmacophore models - Google Patents

In silico screening for shbg binding ligands via pharmacophore models Download PDF

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WO2006081658A1
WO2006081658A1 PCT/CA2006/000130 CA2006000130W WO2006081658A1 WO 2006081658 A1 WO2006081658 A1 WO 2006081658A1 CA 2006000130 W CA2006000130 W CA 2006000130W WO 2006081658 A1 WO2006081658 A1 WO 2006081658A1
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angstroms
distanced
hydrogen bond
site
center
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PCT/CA2006/000130
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French (fr)
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Geoffrey L. Hammond
Zheng Shi
Magid Fallahi
Artem Cherkasov
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The University Of British Columbia
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is related to methods of screening and assaying compounds according to ligand binding as well as pharmaceutical compositions, their use and methods of medical treatment for conditions relating to levels and distribution of sex hormones in a mammal.
  • Sex hormone-binding globulin is a glycoprotein in blood plasma that is produced primarily by the liver [I].
  • Plasma SHBG binds biologically active androgens and estrogens and play a critical role in regulating the access of these sex steroids to their target cells [1-3].
  • SHBG has been reported to interact directly with plasma membranes of cells in some tissues in a ligand-dependent manner, and to thereby stimulate intracellular signalling pathways that alter cell growth and/or function [4].
  • Sex hormone binding globulin regulates the plasma distribution and bioavailability of the endogenous sex steroid hormones that are ligands of SHBG.
  • SHBG Sex hormone binding globulin
  • SHBG with a reduced affinity for sex steroid hormones, or low levels of normal SHBG can lead to increased plasma sex hormone levels.
  • SHBG structure has been described [5-8, 38]. A variety of methods have been used to quantify the serum concentrations of SHBG and assay for the binding affinity [9].
  • the mean serum sex hormone level in healthy premenopausal women is about 84 nmol/L to about 185 nmol/L.
  • Serum SHBG levels are known to be elevated in women treated with oral estrogens, estrogen-containing oral contraceptives, or other estrogen analogs or mimics, as well as in women who are pregnant, hyperthyroid, have chronic liver disease or HIV infection [10].
  • Levels of SHBG and sex steroid hormones may fluctuate in a mammal due to normal biological events such as progressing to sexual maturity, pregnancy, menarche, or menopause, in response to a disease state, or as a consequence or cause of a genetic abnormality.
  • SHBG is known to interact with some sex hormones [11], some plant-derived agents [12], some alkylphenols [13], some flavonoid phytoestrogens [13], some dicyclohexane derivatives [14, 15], some 4-aryl-2,6- dimethylpyridines [16], some hydroxy-polychlorinated- biphenyls [17], some phthalate esters [17], some monoesters[17], some chlorinated pesticides [17], some synthetic estrogens [17], some phytoestrogens [17] and a variety of steroids [18].
  • This invention is based, in part, on the discovery of the common structural features of ligands that bind to SHBG.
  • a method for identifying a ligand for sex hormone binding globulin comprising combining a protein containing a SHBG binding site with a compound and detecting binding of the compound to the protein, said binding being indicative of the compound being said ligand, and wherein the compound comprises pharmacophore A, B or C or a combination thereof, wherein pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.2 angstroms to about 8.5 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 0.3 angstroms to about 5.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 1.0 angstroms to about 5.3
  • pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 0.5 angstroms to about 5.2 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 2.5 angstroms to about 9.0 angstroms from (e), and distanced by about 2.0 angstroms to about 7.6 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 5.7 angstroms to about 12.3 angstroms from (e), distanced by about 4.6 angstroms to about 10.9 angstroms from (f), and distanced by about
  • pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 1.8 angstroms to about 6.6 angstroms from (j); 1) a third hydrophobic/aromatic site distanced by about
  • the method described above wherein the compound comprises pharmacophore A. In another aspect of the present invention, there is provided the method described above, wherein the compound comprises pharmacophore B. In another aspect of the present invention, there is provided the method described above, wherein the compound comprises pharmacophore C.
  • pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.4 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 2.8 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 2.7 angstroms from (c-ii), and distanced by about 2.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site having a center, and: c-i) a first
  • pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 2.0 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 5.7 angstroms from (e), and distanced by about 4.4 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 8.9 angstroms from (e), distanced by about 7.6 angstroms from (f), and distanced by about 3.3 angstroms from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (i-i
  • pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 3.5 angstroms from (j); 1) a third hydrophobic/aromatic site distanced by about 5.8 angstroms from (j), and distanced by about 2.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (m-i), said hydrogen bond donor site distanced by about 2.8 angstroms from (j), distanced by about 6.3 angstroms from (k), and distanced by about 8.5 angstroms from (1); n) a hydrogen bond donor site having a center, and: m-i) a first
  • the protein is SHBG.
  • the SHBG may be human SHBG.
  • methods described above comprising comparing the binding of said compound to said protein to binding of a known SHBG ligand to said protein. This may comprise measuring affinity of the compound for said protein.
  • a method of altering the binding of a sex hormone to SHBG comprising combining SHBG in the presence of the sex hormone with an effective amount of an SHBG ligand comprising a pharmacophore as defined herein, providing that the ligand is not listed in v or 14.
  • the ligand may compete with the sex hormone for binding to SHBG.
  • combining is performed in vivo in a mammal.
  • the mammal may be suffering from a condition characterised by a decrease in availability of the sex hormone or which is amenable to treatment by increasing the availability of the sex hormone.
  • the combining may be in a biological sample from a mammal.
  • the mammal may be a human.
  • a SHBG ligand comprising a pharmacophore as described herein for altering binding of a sex hormone to SHBG, providing that the ligand is not listed in Table 13 or 14.
  • the use may be for preparation of a medicament.
  • the mammal may be a human.
  • composition comprising a pharmaceutically acceptable carrier and an effective amount of at least one SHBG ligand comprising a pharmacophore as described herein, for use in altering binding of a sex hormone to SHBG in a mammal, wherein the ligand is not listed in Table 13 or 14.
  • Ai is selected from the group consisting of: -OH, -SH, -NH 2 . and -NHR, where R is a 2 to 7 atom substituent;
  • An or Ai 2 are independently selected from the group consisting of an alkyl and a substituted alkyl; and, A 3 and A 4 , or A 4 and A 5 , or Ag and A 7 , or A 7 and Ag, or A 9 and A 10 , or A 10 and An may be optionally joined to form a cycloalkyl.
  • D 5 , D 6 , D 7 , D ⁇ , D 9 and D 10 are independently selected from the group consisting of H, an alkyl and a substituted alkyl; and D 5 and D 6 , or D 6 and D 7 , or D 7 and Ds, or Dg and D 9 , or D 9 and D 10 may be optionally joined to form a cycloalkyl structure.
  • a disease selected from the group consisting
  • sex hormone is testosterone or estrogen.
  • a computer implemented method for identifying a candidate ligand for sex hormone binding globulin comprising: I) providing a data structure comprising data for a plurality of chemical structures identifying a functionality for individual sites within each chemical structure according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; II) providing a data structure comprising data for a pharmacophore identifying a functionality for individual sites within the pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and spatial co-ordinates for each site having said functionality; and III) comparing the data structures of (I) and (II) and identifying a chemical structure in (I) matching the pharmacophore of (II); wherein the pharmacophore is: pharmacophore A, B, or C
  • a computer implemented method described above further comprising: IV) providing a data structure comprising data for the candidate ligand comprising said functionality and co-ordinates for sites within the candidate ligand; V) providing a data structure comprising data for a sex hormone binding site of SHBG including functionality of sites according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic and spatial co-ordinates for each site having said functionality; and VI) comparing the data structures of (IV) and (V) to provide a model showing presence or absence of binding of the ligand to the binding site.
  • the data structures (IV) and (V) further comprise a functionality for sites having spatial co-ordinates defining the position of a positive charge.
  • data structures (IV) and (V) further comprise co-ordinates defining size and shape of said ligand and said binding site respectively, wherein said model shows presence or absence of fit of said ligand with said binding site.
  • a computer implemented method described above further comprising: further comprising: VII) obtaining a compound comprising the chemical structure of candidate ligand; VIII) combining the compound with a protein containing the sex hormone binding site; and IX) detecting binding of the compound to the binding site.
  • a computer implemented method described above further comprising: further comprising measuring binding affinity of the compound for the binding site.
  • a data structure embodied in a computer readable medium comprising means for storing a functionality for individual sites within a pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and means for storing spatial co-ordinates for each site having said functionality; wherein the pharmacophore is pharmacophore A, B or C.
  • a data structure embodied in a computer readable medium comprising fields storing a functionality for individual sites within a pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and at least one field for storing spatial co-ordinates for each site having said functionality, wherein the pharmacophore is pharmacophore A, B, or C.
  • the compounds and ligands of the present invention are not the specific steroids described in Tables 13 or the compounds or substances described in Table 14.
  • the compounds and ligands of the present invention are not any steroid, including those listed in Table 14.
  • the term "steroid” as used herein describes a group of compounds which have four fused rings and contain 17 carbon atoms in the fused ring structure. Steroids often have Sterane (1,2-cyclopentanoperhydrophenanthrene) as a core structure, wherein the core structure may be partially or complete hydrogenated and may contain functional substituent groups. Common substituents on the fused ring structure include a C 1O methyl, a Q 3 methyl as well as a C 17 functional group such as a ketone, a hydroxyl or an alkyl.
  • a group of steroid hormones of particular interest are the sex hormones. These are typically produced by the adrenal cortex and/or the gonads. These hormones function generally to promote development and maintenance of the secondary sex characteristics and structures, prepare the female for pregnancy, and aid in development of gametes.
  • Sex hormones are further subdivided into three main categories: estrogens, progestagens and androgens.
  • Estrogens are C ⁇ steroids that always have an oxygenated substituent at C 17 , typically have a phenolic ring in the sterane structure with the hydroxy substituent at C 3 , and typically do not have a C 1O substituent. 17 ⁇ -estradiol is a typical example of an estrogen.
  • Progestagens are C 21 steroids with a en-4-one-3 substituent and a ketone substituent.
  • Progesterone is a typical example of a progestagen.
  • Androgens are Q 9 steroids. Testosterone is a typical example of an androgen.
  • alkyl refers to straight or branched chain unsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms.
  • a substituted alkyl includes an aryl, heterocyclo, heteroaryl, or cycloalkyl substituent
  • said ringed systems are as defined below and thus may in turn have zero to four substituents (preferably 0-2 substituents), also as defined below.
  • Ra, Rb or Rc is an alkyl or alkenyl
  • cycloalkyl refers to fully saturated and partially unsaturated hydrocarbon rings of 3 to 9, preferably 3 to 7 carbon atoms.
  • Figure 1 a) shows graphical depictions of a representative pharmacophore A superimposed with the structure of 5 ⁇ -dihydrotestosterone (DHT). b) shows graphical depictions of a representative pharmacophore B superimposed with the structure of DHT. c) shows graphical depictions of a representative pharmacophore C superimposed with the structure of DHT.
  • DHT 5 ⁇ -dihydrotestosterone
  • Figure 2 a) illustrates the distances between the individual features of an embodiment of pharmacophore A.
  • b) illustrates the angles between the individual features of the embodiment in 2(a).
  • c) illustrates the distances between the individual features of an embodiment of pharmacophore B.
  • d) illustrates the angles between the individual features of the embodiment in 2(c).
  • e) illustrates the distances between the individual features of an embodiment of pharmacophore C.
  • f) illustrates the angles between the individual features of the embodiment in 2(e).
  • Figure 3 shows the configuration of the Artificial Neural Network (ANN) containing 28 input, 8 hidden and 1 output nodes, used for ranking SHBG ligands.
  • ANN Artificial Neural Network
  • Figure 4 depicts the structure of a portion of human SHBG containing DHT (a) or estradiol (b) in the steroid-binding pocket.
  • Figure 5 shows the relationship plot between experimental association constant (LogK a ) and the consensus docking score.
  • Figure 6 shows a superimposition of DHT molecules docked into SHBG using Glide software (light) with bound DHT configuration defined from the crystal structures (dark).
  • Figure 7 shows the distribution of the docking consensus score for pharmacophore-identified compounds and randomly derived molecular structures.
  • Figure 8 shows the displacement curves for test compounds used in the competition assay to determine the relative binding affinities of human SHBG ligands.
  • the amount of [ 3 H] DHT bound to SHBG in the presence of increasing concentrations of competitor ligands (B) is expressed as a percentage of the amount of [ 3 H] DHT bound to SHBG in the absence of competitor ligand (Bo).
  • Figure 9 shows the structural alignment of a compound l-(2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)ethanone (5597) with other SHBG ligands.
  • acophore A Range of possible (x,y,z) co-ordinates for each feature
  • Pharmacophore B Range of possible (x,y,z) co-ordinates for each feature
  • Pharmacophore C Range of possible (x,y,z) co-ordinates for each feature
  • Hydrogen bond acceptor/donor, acceptor and donor features are features that are capable of: i) accepting a hydrogen bond from hydrogen bond donor, ii) donating a hydrogen bond to a hydrogen bond acceptor or iii) capable of both.
  • a hydrogen bond is formed by three atoms: one hydrogen atom and two electronegative atoms. The hydrogen atom is covalently bonded to one of the electronegative atoms (the hydrogen bond donor), the other electronegative atom is the hydrogen bond acceptor. The two electronegative atoms take up some electron density from the hydrogen atom, and as a result, each electronegative atom carries partial negative charge and the hydrogen atom carries partial positive charge. The hydrogen atom and the hydrogen bond acceptor can then have attractive interactions. For a review of hydrogen bonding, see [19].
  • Hydrogen bond donors include -OH, -SH, -NH 2 , -NHR, where R is a 2 to 7 atom substituent.
  • Halogens include chlorine, bromine, fluorine and iodine.
  • Aromatic structures are typically planar, cyclic structures where all of the atoms in the ring or rings are sp2 and the structure has a free "p" orbital in which Pi electrons can travel through so as to be distributed throughout the structure.
  • Aromatic structures are typically resonance stabilised and have 4n + 2Pi electrons, wherein n is any integer. Benzene rings, naphthyl rings, benzene rings substituted with a heteroatom, and naphthyl rings substituted with a heteroatom are typical examples of aromatic structures.
  • Hydrophobic structures are non-polar structures that do not readily interact with or dissolve in water. Typical examples of hydrophobic structures include unsubstituted alkanes, alkyls, and alkenyls. Often aromatic structures are also hydrophobic structures.
  • Sites of substitution unrestrained by the pharmacophore may be found on aromatic or aliphatic or heteroorganic ring structures and double bond structures of the parent molecule, as well as other sites. Many of these sites are not as restricted by the pharmacophores as are sites of hydrogen bond donor/acceptors.
  • a projection point is a feature on a pharmacophore that restricts or defines the outer extent of a hydrogen bond donor/acceptors site, a hydrogen bond donor site or a hydrogen bond acceptor site in one direction from the centre.
  • a plurality of projection points provides information concerning both shape and size of the particular a hydrogen bond donor/acceptor site, a hydrogen bond donor site or a hydrogen bond acceptor site to which the projection point is associated.
  • the pharmacophores described herein can be used to screen electronic libraries of compounds using techniques known to one skilled in the art (see, for example, Y. C. Martin, 3D Database Searching in Drug Design, J Med. Chem. 35, 2145(1992); A. C. Good and J. S. Mason, Three Dimensional Structure Satabase searches, Reviews in Comp. Chem. 7, 67 (1996); Guner, O. F. Pharmacophore perception, Development, and Use in Drug Design, IUL Biotechnology Series; International University Line: La Jolla, CA, (2000); Klebe, G.
  • Structural data on a panel of compounds may be selected from multiple sources, including combinatorial libraries, compound collections and other sources. The structural data is most frequently presented in a database format. Examples of suitable databases include Dictionary of Natural Products DNP (www.crcpress.com), Bioactive Natural Products (Szenzor Co., Hungary), BioScreenNP files (www.ibscreen.com/natural.shtml), Marine Natural Product Database MNPD [Lei, J., Zhou, J. A Marine Natural Product Database. /. Chem. Inf. Comp.
  • QSAR quantitative structure-activity relationship
  • ANN artificial neural networks
  • virtual docking techniques There are many ways to apply these techniques and it is within the understanding of one skilled in the art to apply these techniques appropriately in order to prioritise the hits obtained by screening an electronic library using a particular pharmacophore.
  • QSAR quantitative structure-activity relationship
  • ANN artificial neural networks
  • virtual docking techniques There are many ways to apply these techniques and it is within the understanding of one skilled in the art to apply these techniques appropriately in order to prioritise the hits obtained by screening an electronic library using a particular pharmacophore.
  • pharmacophore A is preferably for identifying steroidal ligands, but will often identify less ligands than Pharmacophores B and C.
  • Pharmacophore C is preferable for identifying non-steroidal ligands and will often identify the largest number of ligands, and in rare cases, may also identify some false positive hits that will require wet assaying to identify as false positives.
  • Pharmacophores B will identify steroidal ligands and non-steroidal ligands, and often identify more ligands than pharmacophore A, but less than pharmacophore C.
  • Each of pharmacophore A, B and C are capable of identifying ligands that the other pharmacophores would not identify. Using combinations of the pharmacophores will identify ligands that have the properties associated with each of the pharmacophores used. Ligands identified using combinations of the pharmacophores will likely have features common to steroidal ligands and non-steroidal ligands. Identification of SHBG ligands with no intrinsic biological properties of their own (or with properties selected for a particular effect) which block or compete for sex hormone binding represent a means of altering the role of SHBG in the body. For example, use of such ligands which have no intrinsic steroidal activity may enhance the bioavailability and activity of endogenous sex hormones.
  • Known ligands of SHBG [18] were used to develop pharmacophore models of the SHBG steroid-binding site. These pharmacophores were then used to screen an electronic library of compounds. This led to identification of a smaller set of structures that met the stringent structural requirements imposed by these pharmacophores.
  • the known set of SHBG ligands were then used to train a QSAR model algorithm to differentiate known SHBG ligands from other structures. The resulting structure-activity model was then applied to rank the pharmacophore-identified compounds for their potential binding affinity toward SHBG.
  • the QSAR model assigned very significant network outputs above 0.5 to four of the most active compounds which produce a >35% reduction of [ 3 H] DHT binding to SHBG in the screening assay (compounds l,l'-Spirobi[indane-5,6-diol], 3,3,3',3'-tetramethyl- (696), 4-[5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]phenol (2593), 4-[5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]-2-methoxy-phenol (2623), 4-[5-(hydroxymethyl)-2-methyl-7-oxabicyclo[3.3.
  • Assays that may be used to test the binding affinity of a particular compound to SHBG are known by those of one of skill in the art. These assays include competition assays using rat prostate androgen receptor [16], competition assays using rat, rabbit, money or human epididymal androgen binding protein [16], competition assays using human sex hormone binding globulin, [16] screening assays based on the ammonium sulfate precipitation method [17], competitive binding assays in diluted human pregnancy serum [17], and charcoal assays using varying concentrations of the testing compound and tritiated DHT [12].
  • a detailed example of a particular binding assay that may be used is described in Example 4.
  • assays that may be used to determine the binding affinity of a particular ligand to SHBG may be found in Jury, et al., Journal of Steriod Biochemistry & Molecular Biology, 75 (2000) 167-176; Winneker, et al, J. Med., Chem. 33 (1990), 129-132; Rousseau, G.G. et al, J Steroid Biochem. 31 (1988), 691-697; Rousseau, G.G., et al, Nature. 284 (1980), 458-459; and Schottner, M., etal., J Nat Products. 61 (1998), 119-121.
  • the binding affinities of selected testing candidates as determined by a competitive displacement assay described in Example 4 are shown in Figure 8.
  • the affinity of compounds 2623 and 5597 exceed those of other synthetic and natural non-steroidal compounds with endocrine disrupting properties, which typically bind to SHBG steroid-binding site with association constants in the range of 0.02 x 10 5 M "1 to 8 x 10 5 M '1 [12, 13, 20].
  • Figure 9a shows a superimposition of the molecular structures of 5597 and (-)-3, 4-divanyllyltetrahydrofuran, a natural non-steroidal lignan with a high association constant for SHBG [12].
  • the overall shape of these molecules is similar, and critical chemical groups required for SHBG binding are positioned closely in space.
  • the ability of compound 5597 and others to displace DHT and estradiol from SHBG indicates that these non-steroidal compounds identified interact with the SHBG steroid-binding pocket.
  • Figure 9b shows the structure of 5597 docked into the active site of SHBG and superimposed against bound DHT. In this orientation, 5597 reproduces all critical binding features of a sex steroid.
  • Two other non-steroidal ligands of SHBG are structurally very similar but differ in their binding affinities.
  • the main structural difference between them is the presence of a methoxy-group adjacent to a hydroxyl group on a phenolic ring structure.
  • a similar difference is also observed in the relative binding affinities of estradiol and 2-methoxy-estradiol for human SHBG [18], suggesting that the phenolic ring structures of compounds 2623 and 2593 reside within the SHBG steroid-binding site in the same orientation as ring A of an estrogen molecule.
  • the compounds identified using techniques described herein are suitable for treating diseases that are characterised by undesirable levels, high or low, of sex hormone in the plasma.
  • Such diseases include, osteoporosis, female sexual dysfunction, endometriosis, anorexia nervosa, androgen producing ovarian cancer hyperthyroidism, hypogonadism (males), androgen insensitivity deficiency, alcoholic hepatic cirrhosis (males), and primary biliary cirrhosis (females), Klinefelter's syndrome, teratogenic disorders, cervical dysplasia, ovarian dysfunction, male or female infertility, sexual or erectile dysfunction, decreased libido, vaginal dryness, thinning of the vaginal wall, menopause symptoms including hot flashes, osteopenia, diabetes, hyperglycemia, hyperglyceridemia, hypercholesterolemia, hypertension, atherosclerosis, cardiovascular disorders and disease, obesity, neurological dysfunction, Alzheimer's disease, dementia and cataracts.
  • ligands that have been identified using the pharmacophores described herein are capable of binding to sex hormone binding globulin (SHBG) and consequently competing with sex hormones for binding to SHBG.
  • SHBG sex hormone binding globulin
  • ligands identified herein can increase the concentration of available sex hormone in a mammal.
  • Ligands that bind to SHBG may also be coupled to or synthesised to include known labelling moieties such as radio-isotopes and then used for imaging. Such labelled compounds are useful for imaging certain types of cancer, particularly estrogen secreting cancers. In these types of cancer, the labelled ligand is sequestered at the site of the cancer because the estradiol secreted by the cancer displaces the labelled ligand more frequently at the cancer site. High concentration of estradiol at these cancer sites results in a higher concentration of labelled ligand being displaced at the cancer site. Examples of labelling ligands of SHBG can be found in Seimbille et al. Steroids 67, (2002) 765-775.
  • a desired moiety such as a cytotoxic compound, for example cisplatin or other anti-cancer agents could be coupled to a SHBG ligand.
  • Such moieties are delivered and deposited at a desired site such as an estrogen secreting cancer.
  • Cytotoxic moieties coupled to ligands in this way could be more effective as therapeutics by minimising side effects from the cytotoxic agent on the patient since more of the cytotoxic agent would be delivered to the desired site and less cytotoxic compound would be delivered to undesired sites.
  • suitable target cancers include endometrial cancer, cervical cancer, uterine cancer and breast cancer. Labelling and coupling to SHBG ligands as described above could be applied to areas and tissues having a high androgen concentration, for example androgen secreting ovarian tumors, as well as areas and tissues with high estrogen concentrations.
  • Substituent group A 1 functions generally as a hydrogen donor group.
  • Hydrogen bond donor groups include -OH, -SH, -NH 2 , -NHR, where R is a 2 to 7 atom substituent.
  • Substituent group A 7 functions generally as a hydrogen acceptor group.
  • Substituents groups A 2 , A 3 , A 4 , A 5 , A $ , As, A 9 , Aio, An or Ai 2 may be alkyl groups or substituted alkyl groups.
  • Substituent groups A 3 and A 4 , or A 4 and A 5 , or A 6 and A 7 , or A 7 and A ⁇ , or A 9 and Aio, or Aio and An may be joined as cycloalky
  • Synthesis of compound 5597 and derivatives depicted generally in Formula I may be prepared according to methods described in US 6677350, US 5208343, Kenkyu Hokuku - Sen'i Kobunshi Zairyo Kenkyusho 155:45-8 (1987), J. Appl. Polym. Sci 32:4127-35 (1986), Bulletin de Ia Societe Chimique de France (1961) 584-6, JP 58025303, JP54117482 and DE 1921348, and other methods known in the art.
  • Substituent groups Ei or E 2 function generally as hydrogen acceptor groups.
  • Substituent groups E 7 or Es function generally as hydrogen donor groups.
  • Hydrogen bond donor groups include -OH, -SH, -NH 2 , -NHR, where R is a 2 to 7 atom substituent.
  • Substituent groups Ej, E 2 , E 3 , E 4 , E 5 , Eg, E 7 Eg, E 9 , E 10 or En may be alkyl groups or substituted alkyl groups.
  • Substituent groups E 4 and E 5 , or E 5 and Eg or E 9 and E 10 , or Eio and En may be joined as cycloalkyl groups.
  • Synthesis of compound 5636 and derivatives as depicted generally in Formula II may be prepared according to methods described in US 6677350, US 5208343, Kenkyu Hokuku - Sen'i Kobunshi Zairyo Kenkyusho 155:45-8 (1987), J. Appl. Polym. Sci 32:4127-35 (1986), Bulletin de Ia Societe Chimique de France (1961) 584-6, JP 58025303, JP54117482 and DE 1921348 and other methods known in the art.
  • Substituent groups B 5 , Bg or B 7 function generally as hydrogen donor groups.
  • Hydrogen bond donor groups include -OH, -SH, -NH 2 , -NHR, where R is a 2 to 7 atom substituent.
  • Substituent groups Bi, B 2 or B 3 functional generally as hydrogen acceptor groups.
  • Substituent groups Bi, B 2 , B 3 , B 4 , B 5 , B 6 or B 7 may be alkyl groups or substituted alkyl groups.
  • Substituent groups B 5 and Be or B 6 and B 7 may be joined as cycloalkyl groups.
  • Synthesis of compound 6767 or 8590 and derivatives depicted generally as Formula III may be prepared according to methods described in J. Appl. Microbiol. 91:1118-1130 (2001), Tx. J. Science 17:340-4 (1965), Khimiya Geterotsiklicheskikh Soedinenii 2:150-4 (1970) and GB 1044608, and other methods known in the art.
  • Substituent groups Di and D 2 , D 3 and D 4 function generally as hydrogen donor and hydrogen acceptor groups.
  • Hydrogen bond donors include -OH, -SH, -NH 2 , -NHR, where R is a 2 to 7 atom substituent.
  • Substituent groups D 5 , Dg, D 7 , Dg, D 9 and Djo may be alkyl groups or substituted alkyl groups.
  • Substituent groups D 5 and D ⁇ , or Dg and D 7 , or D 7 and Dg, or Dg and D 9 , or D 9 and D 10 may be joined as cycloalkyl structures.
  • Synthesis of compound 696 and derivatives depicted generally as Formula IV may be prepared according to methods described in JP 09110771, Polym. Mater. Sci. Eng. 70:378-9 (1993), JP 03148232, JP 02286642, ip 03386641, JP 02248954, EP 342035, EP 307951, Bur. Polym. J. 15(7):631-8 (1979), FR 2322161, Izv. Akad, Nauk. SSSR. Ser. Khim- (12):2808-10 (1973) and Tetrahedron Lett. (34):3707:10 (1968), and other methods known in the art.
  • Substituent groups Fi or F 2 function generally as hydrogen donor groups.
  • Hydrogen bond donor groups include -OH, -SH, -NH 2 , -NHR, where R is a 2 to 7 atom substituent.
  • Substituent groups F 3 or F 4 function generally as hydrogen bond acceptor groups.
  • Substituent groups Fi, F 2 , F 3 , F 4 , F 5 , F 6 , F 7 , F$ or F 9 may be alkyl groups or substituted alkyl groups.
  • the efficacy of the compounds described herein may be measured by performing an SHBG binding assay to determine the binding affinity of a particular ligand.
  • a particular ligand can be selected based on its binding affinity for SHBG. For example, if the disease is characterized by low levels of bio-available sex hormone, such as female sexual dysfunction or osteoporosis, then a ligand with a high binding affinity for SHBG will be chosen so that it occupies a ligand binding pocket of SHBG for longer periods, thereby preventing the bio-available androgens from binding to SHBG and encouraging more bio-available androgen hi the plasma.
  • a lower binding affinity for SHBG may be desired so that it becomes easier for the imaging agent to be displaced by a sex hormone.
  • a ligand to be labeled may be selected so that is has an affinity that is moderately or slightly weaker than a sex hormone that is produced by the target cancer tissue.
  • Such a labeled ligand will stay bound to SHBG with a maximum efficiency until it contacts a sex hormone that has a higher binding affinity for SHBG. This is mostly likely to occur at the cancer site and displacement of the ligand will result in deposition of the imaging agent in the cancer site, thereby providing a better image.
  • a lower binding affinity for SHBG may be desired so that it becomes easier for the cytotoxic moiety to be displaced by a sex hormone, for example a sex hormone being produced by the target cancer.
  • a ligand to be coupled may be selected so that is has an affinity that is moderately or slightly weaker than a sex hormone that is produced by the target cancer tissue.
  • Such a coupled ligand will stay bound to SHBG with a maximum efficiency until it contacts a sex hormone that has a higher binding affinity for SHBG. This is mostly likely to occur at the cancer site and displacement of the ligand will result in deposition of the cytotoxic moiety in the cancer site, thereby providing improved therapy with less side effects.
  • compositions in accordance with this invention may comprise a salt of such a compound, preferably a physiologically acceptable salt, which are known in the art.
  • Pharmaceutical preparations will typically comprise one or more carriers acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers are those known in the art for use in such modes of administration.
  • Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner.
  • a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non- water soluble compounds.
  • enteral administration the compound may be administered in a tablet, capsule or dissolved in liquid form.
  • the tablet or capsule may be enteric coated, or in a formulation for sustained release.
  • Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, gels, hydrogels, or solutions which can be used topically or locally to administer a compound.
  • a sustained release patch or implant may be employed to provide release over a prolonged period of time.
  • Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for modulatory compounds include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • Pharmacophore A was developed from the structures of 5-alpha-dihydrotestosterone (DHT) and its close derivatives ( Figure Ia, 2a, 2b).
  • This pharmacophore model included nine major features (Table 4): two hydrophobic/aromatic sites (F2, F3), one hydrogen bond donor/acceptor point (F6) with three donor/acceptor projection points (F7, F8 and F9) and one hydrogen bond acceptor (Fl) with two acceptor projection points (F4 and F5).
  • Pharmacophore B was generated by aligning the crystallographic configurations of DHT and estradiol inside the steroid-binding pocket of SHBG, and it utilised seven basic features (Table 5): three hydrophobic/aromatic centers (F3, F5, F6), one H acceptor (Fl) and one H-bond donor (F2), with two donor projection points (F4, F7) ( Figure Ib, 2c, 2d).
  • Pharmacophore C relied on the flexible alignment of a large number of known ligands of human SHBG [18], which resulted in the identification of the following common features (Table 6): three hydrophobic/aromatic centers, one hydrogen donor site with two donor projection points and one H-bond acceptor with two projection points (Figure Ic, 2e, 2f).
  • True hit rate number of true hits / total number of hits
  • False hit rate number of false hits / total number of hits
  • Recovery rate number of true hits / total number of true hits
  • Oclccc2c(cl)CC Cl[C@H](CC[C@H](O)[C@ @](C)
  • N C(Ncl[nHO]c(C)cc([nHO]l)C)Nclcccc(C)cl
  • OCC(O)C 1 OCC(O)C 1 (OC2(C)CCC3C(C)(C)C(O)CCC3(C)C2CC
  • the MOE package has been used to optimise all molecular structures [22].
  • the 'inductive' descriptors have been computed by the MOE custom written scripts for all compounds studied.
  • the ANN training has been created and used by the SNNS package [24].
  • a learning rate of 0.8 and a learning update threshold of 0.2 were used, while the input patterns were shuffled.
  • the number of hidden nodes varied from 2 to 20, and 20 independent training and testing runs were conducted for each configuration.
  • the corresponding statistical parameters featured in Tables 10a and 10b represent the values averaged over those 20 training/testing runs.
  • the identified 105 non-steroidal chemicals were prioritised for experimental testing by using a QSAR approach utilising inductive molecular descriptors [25-27, 28-31].
  • These descriptors are a novel and distinct group of QSAR descriptors that cover a broad range of bound atoms and molecules whose properties vary in relation to their size; polarizability; electronegativity; compactness; mutual inductive and steric influence, and distribution of electronic density.
  • Inductive descriptors were used in combination with the method of Artificial Neural Networks (ANN) to rank the 105 non-steroidal compounds selected according to their potential binding-affinity to human SHBG.
  • ANN Artificial Neural Networks
  • To build a predictive QSAR model a set of more than 70 compounds known to interact with SHBG (entries 1-78 of Table 8) were assembled, and complied a set of about two hundred chemicals [32] with unknown affinities to SHBG as negative controls for the model (entries 79-282 of Table 8). For all 282 molecules, 28 independent inductive QSAR descriptors, that are characterised in greater detail in Table 9, were calculated.
  • the ANN-based binary QSAR model created was applied to the 105 molecules identified previously by the pharmacophore model-based search for potential SHBG ligands (entries 283-388 of Table 8).
  • the molecular structures of all 105 molecules were processed to calculate 28 inductive descriptors that were then passed through the pre-trained ANN.
  • the resulting network outputs have been assembled in Table 8 and it can be seen that some candidate molecules are ranked very highly by the model.
  • the corresponding top-ranked substances represent very good candidate ligands of human SHBG, and 22 compounds were selected (molecules from Table 8 with network outputs above 0.1) for purchasing and testing.
  • a ligand-protein docking procedure was applied to all 105 pharmacophore-identified compounds to support the lead selection by the QSAR model and to expand the set of selected compounds.
  • the Maestro suite [33] was used to prepare protein structures for docking, which was conducted using the Glide 2.7 program [34] with default settings.
  • the protein charges were assigned by the MMFF94 molecular mechanics force field method [35] and the binding pocket of SHBG was identified as 6.5A surrounding the natural ligand DHT from the crystal structure 1D2S.
  • a consensus docking strategy was developed utilising four SHBG protein structures - PDB entries 1D2S (co-crystallised with DHT), 1F5F, IKDM and ILHU (co-crystallised with estradiol). All protein structures were pre-processed for docking by removing water molecules and reconstructing hydrogen atoms.
  • the Glide 2.7 program [34] was used to fit 32 SHBG binders with experimentally determined association constants (correspond to entries 1-8, 10-12, 14-16, 18, 19-21, 28, 32, 33, 38, 41, 44, 46, 51, 53 from Table 8) into the four different crystal structures of SHBG. Based on the docking results for all four structures, an averaged consensus score was derived, which was plotted in Figure 5 against known ligand-SHBG association constants (K a ) taken from the literature [18]. Figure 5 shows the r value for the correlation is above 0.81, indicating that the docking protocol reproduced the experimental protein binding affinities with accuracy.
  • Figure 6 illustrates the docked DHT structure, and shows that it is closely aligned with the known orientation of DHT within the crystal structure (1D2S) of the SHBG steroid-binding site. It was also established that, on average, a compound demonstrates good binding affinity to SHBG when the corresponding docking score is below the -7.5 threshold.
  • Figure 7 illustrates the distribution of the consensus docking scores among known SHBG binders and compounds with no known SHBG binding affinity (molecules 82-151 from Table 8 which have been docked as negative controls).
  • An established competitive ligand binding assay was used to determine the binding affinities of test compounds to human SHBG in relation to those of testosterone and estradiol [43].
  • the assay involved mixing 100 ⁇ l aliquots of diluted (1:200) human pregnancy serum containing approximately 1 nM SHBG, which was pre-treated with dextran-coated charcoal (DCC) to remove endogenous steroid ligand, with 100 ⁇ l tritium labelled DHT ([ 3 H] DHT) at 10 nM as labelled ligand.
  • DCC dextran-coated charcoal
  • the 29 compounds selected by the QSAR ranking and virtual docking experiments were screened for their ability to interact with the SHBG steroid-binding site.
  • the screening assay involved a modification of an established competitive steroid ligand-binding assay that employs tritium-labelled DHT ([ 3 H] DHT) as the radio-labelled ligand as described above.
  • the initial screen of compounds was conducted at a single high concentration (approximately 200 ⁇ M).
  • MOE Molecular Operational Environment; Version 2003.10, Chemical Computation Group Inc., Montreal, Canada, 2004.

Abstract

Provides methods for identifying and using ligands for the sex hormone binding site of sex hormone binding globulin (SHBG) as well as such novel ligands are provided. Also provided are compositions comprising such ligands for use in modifying sex hormone binding to SHBG. Further provided is a computer implemented method for identifying SHBG ligands and data structures embodied in computer readable media useful in such a method. SHBG ligands comprise a defined pharmacophore.

Description

in silico Screening for SHBG Binding Ligands via Pharmacophore Models
Field of the Invention:
The present invention is related to methods of screening and assaying compounds according to ligand binding as well as pharmaceutical compositions, their use and methods of medical treatment for conditions relating to levels and distribution of sex hormones in a mammal.
Background of the Invention:
Sex hormone-binding globulin (SHBG) is a glycoprotein in blood plasma that is produced primarily by the liver [I]. Plasma SHBG binds biologically active androgens and estrogens and play a critical role in regulating the access of these sex steroids to their target cells [1-3]. In addition to binding steroids, SHBG has been reported to interact directly with plasma membranes of cells in some tissues in a ligand-dependent manner, and to thereby stimulate intracellular signalling pathways that alter cell growth and/or function [4].
Sex hormone binding globulin (SHBG) regulates the plasma distribution and bioavailability of the endogenous sex steroid hormones that are ligands of SHBG. Excess plasma SHBG, or SHBG with an abnormal binding affinity for plasma steroids, can lead to reduced plasma sex hormone levels. SHBG with a reduced affinity for sex steroid hormones, or low levels of normal SHBG can lead to increased plasma sex hormone levels.
SHBG structure has been described [5-8, 38]. A variety of methods have been used to quantify the serum concentrations of SHBG and assay for the binding affinity [9]. The mean serum sex hormone level in healthy premenopausal women is about 84 nmol/L to about 185 nmol/L. Serum SHBG levels are known to be elevated in women treated with oral estrogens, estrogen-containing oral contraceptives, or other estrogen analogs or mimics, as well as in women who are pregnant, hyperthyroid, have chronic liver disease or HIV infection [10].
Levels of SHBG and sex steroid hormones may fluctuate in a mammal due to normal biological events such as progressing to sexual maturity, pregnancy, menarche, or menopause, in response to a disease state, or as a consequence or cause of a genetic abnormality. Diseases, or the health problems associated with them, related to aberrant levels or function of SHBG include osteoporosis, female sexual dysfunction, endometriosis, anorexia nervosa, androgen producing ovarian cancer hyperthyroidism, hypogonadism (males), androgen insensitivity deficiency, alcoholic hepatic cirrhosis (males), and primary biliary cirrhosis (females), Klinefelter' s syndrome, teratogenic disorders, cervical dysplasia, ovarian dysfunction, male or female infertility, sexual or erectile dysfunction, decreased libido, vaginal dryness, thinning of the vaginal wall, menopause symptoms including hot flashes, osteopenia, diabetes, hyperglycemia, hyperglyceridemia, hypercholesterolemia, hypertension, atherosclerosis, cardiovascular disorders and disease, obesity, neurological dysfunction, Alzheimer's disease, dementia and cataracts.
SHBG is known to interact with some sex hormones [11], some plant-derived agents [12], some alkylphenols [13], some flavonoid phytoestrogens [13], some dicyclohexane derivatives [14, 15], some 4-aryl-2,6- dimethylpyridines [16], some hydroxy-polychlorinated- biphenyls [17], some phthalate esters [17], some monoesters[17], some chlorinated pesticides [17], some synthetic estrogens [17], some phytoestrogens [17] and a variety of steroids [18].
Summary of the Invention:
This invention is based, in part, on the discovery of the common structural features of ligands that bind to SHBG.
In one aspect of the present invention, there is provided a method for identifying a ligand for sex hormone binding globulin (SHBG), the method comprising combining a protein containing a SHBG binding site with a compound and detecting binding of the compound to the protein, said binding being indicative of the compound being said ligand, and wherein the compound comprises pharmacophore A, B or C or a combination thereof, wherein pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.2 angstroms to about 8.5 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 0.3 angstroms to about 5.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 1.0 angstroms to about 5.3 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 1.0 angstroms to about 6.2 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 0.9 angstroms to about 5.4 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 1.0 angstroms to about 6.1 angstroms from (c-i), and distanced by about 1.0 angstroms to about 5.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site center being distanced by about 5.4 angstroms to about 10.5 angstroms from (a), and distanced by about 1.2 angstroms to about 5.7 angstroms from (b); d) a hydrogen bond acceptor site having a center, and: d-i) a first acceptor projection point distanced by about 0.8 angstroms to about 5.4 angstroms from the hydrogen bond acceptor site center; and d-ii) a second acceptor projection point distanced by about
1.0 angstroms to about 5.3 angstroms from the hydrogen bond acceptor site center, and distanced by about 0.8 angstroms to about 5.9 angstroms from (d-i), said hydrogen bond acceptor site center being distanced by about 0.8 angstroms to about 7.0 angstroms from (a), distanced by about 6.0 angstroms to about 12.1 angstroms from (b), and distanced by about
8.1 angstroms to about 14.0 angstroms from the hydrogen bond donor/acceptor site center; pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 0.5 angstroms to about 5.2 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 2.5 angstroms to about 9.0 angstroms from (e), and distanced by about 2.0 angstroms to about 7.6 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 5.7 angstroms to about 12.3 angstroms from (e), distanced by about 4.6 angstroms to about 10.9 angstroms from (f), and distanced by about
1.2 angstroms to about 6.7 angstroms from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 0.3 angstroms to about
5.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about 0.4 angstroms to about 5.1 angstroms from the hydrogen bond donor site center, and distanced by about 0.6 angstroms to about 5.9 angstroms from i-i), said hydrogen bond donor site center being distanced by about 1.4 angstroms to about 6.4 angstroms from (e), distanced by about 1.4 angstroms to about 6.6 angstroms from (f), distanced by about 4.9 angstroms to about 10.9 angstroms from (g), and distanced by about 7.9 angstroms to about 14.1 angstroms from the hydrogen bond acceptor site center; pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 1.8 angstroms to about 6.6 angstroms from (j); 1) a third hydrophobic/aromatic site distanced by about 3.9 angstroms to about 8.6 angstroms from (j), and distanced by about 0.7 angstroms to about 5.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 0.4 angstroms to about 5.1 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about 1.1 angstroms to about 5.3 angstroms from the hydrogen bond donor site center, and distanced by about 0.8 angstroms to about
6.0 angstroms from (m-i), said hydrogen bond donor site center being distanced by about
1.1 angstroms to about 6.2 angstroms from (j), distanced by about 3.6 angstroms to about 9.4 angstroms from (k), and distanced by about 6.2 angstroms to about 11.4 angstroms from (1); n) a hydrogen bond acceptor site having a center, and: n-i) a first acceptor projection point distanced by about 0.7 angstroms to about 5.1 angstroms from the hydrogen bond acceptor site center; and n-ii) a second acceptor projection point distanced by about 1.0 angstroms to about 5.4 angstroms from the hydrogen bond acceptor site center, and distanced by about 1.0 angstroms to about 5.5 angstroms from (n-i), said hydrogen bond acceptor site center being distanced by about 6.2 angstroms to about 11.0 angstroms from (j), distanced by about
3.0 angstroms to about 7.7 angstroms from (k), distanced by about 1.0 angstroms to about
5.7 angstroms from (1), and distanced by about 8.6 angstroms to about 13.8 angstroms from the hydrogen bond donor site center.
In another aspect of the present invention, there is provided the method described above, wherein the compound comprises pharmacophore A. In another aspect of the present invention, there is provided the method described above, wherein the compound comprises pharmacophore B. In another aspect of the present invention, there is provided the method described above, wherein the compound comprises pharmacophore C.
In another aspect of the present invention, there is provided the method described above, wherein pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.4 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 2.8 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 2.7 angstroms from (c-ii), and distanced by about 2.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site center being distanced by about 7.6 angstroms from (a), and distanced by about 2.4 angstroms from (b); d) a hydrogen bond acceptor site having a center, and: d-i) a first acceptor projection point distanced by about
2.1 angstroms from the hydrogen bond acceptor site center; and d-ii) a second acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond acceptor site center, and distanced by about 2.6 angstroms from (d-i), said hydrogen bond acceptor site center being distanced by about 3.6 angstroms from (a), distanced by about 8.9 angstroms from (b), and distanced by about 10.9 angstroms from the hydrogen bond donor/acceptor site center.
In another aspect of the present invention, there is provided the method described above, wherein pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 2.0 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 5.7 angstroms from (e), and distanced by about 4.4 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 8.9 angstroms from (e), distanced by about 7.6 angstroms from (f), and distanced by about 3.3 angstroms from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (i-i), said hydrogen bond donor site center being distanced by about 3.1 angstroms from (e), distanced by about 3.3 angstroms from (f), distanced by about 7.7 angstroms from (g), and distanced by about 10.9 angstroms from the hydrogen bond acceptor site.
In another aspect of the present invention, there is provided the method described above, wherein pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 3.5 angstroms from (j); 1) a third hydrophobic/aromatic site distanced by about 5.8 angstroms from (j), and distanced by about 2.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (m-i), said hydrogen bond donor site distanced by about 2.8 angstroms from (j), distanced by about 6.3 angstroms from (k), and distanced by about 8.5 angstroms from (1); n) a hydrogen bond acceptor site having a center: n-i) a first acceptor projection point distanced by about 1.9 angstroms from the hydrogen bond acceptor site center; and n-ii) a second acceptor projection point distanced by about 2.0 angstroms from the hydrogen bond acceptor site center, and distanced by about 2.2 angstroms from (n-i), said hydrogen bond acceptor site distanced by about 8.3 angstroms from (j), distanced by about 4.9 angstroms from (k), distanced by about 2.6 angstroms from (1), and distanced by about 11.0 angstroms from the hydrogen bond donor site center.
In another aspect of the present invention, there is provided methods described above, wherein the protein is SHBG. The SHBG may be human SHBG.
In another aspect of the present invention, there is provided methods described above comprising comparing the binding of said compound to said protein to binding of a known SHBG ligand to said protein. This may comprise measuring affinity of the compound for said protein. In another aspect of the present invention, there is provided a method of altering the binding of a sex hormone to SHBG comprising combining SHBG in the presence of the sex hormone with an effective amount of an SHBG ligand comprising a pharmacophore as defined herein, providing that the ligand is not listed in v or 14. The ligand may compete with the sex hormone for binding to SHBG.
In another aspect of the present invention, there is provided methods described above wherein said combining is performed in vivo in a mammal. The mammal may be suffering from a condition characterised by a decrease in availability of the sex hormone or which is amenable to treatment by increasing the availability of the sex hormone. The combining may be in a biological sample from a mammal. The mammal may be a human.
In another aspect of the present invention, there is provided the use of a SHBG ligand comprising a pharmacophore as described herein for altering binding of a sex hormone to SHBG, providing that the ligand is not listed in Table 13 or 14. The use may be for preparation of a medicament.
In another aspect of the present invention, there is provided the uses described above for treatment of a condition in a mammal characterised by a decrease in availability of the sex hormone or which is amenable to treatment by increasing the availability of the sex hormone. The mammal may be a human.
In another aspect of the present invention, there is provided a composition comprising a pharmaceutically acceptable carrier and an effective amount of at least one SHBG ligand comprising a pharmacophore as described herein, for use in altering binding of a sex hormone to SHBG in a mammal, wherein the ligand is not listed in Table 13 or 14.
In another aspect of the present invention, there is provided a method, use or composition described above, or a ligand identified by a method described above, wherein the ligand has the structure of Formula I:
Figure imgf000009_0001
Formula I wherein Ai is selected from the group consisting of: -OH, -SH, -NH2. and -NHR, where R is a 2 to 7 atom substituent; A7 is selected from the group consisting of: =0, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, and ether-linked aliphatic rings having between 3 and 7 members; A2, A3, A4, A5, A6, Ag, A9, A10, An or Ai2 are independently selected from the group consisting of an alkyl and a substituted alkyl; and, A3 and A4, or A4 and A5, or Ag and A7, or A7 and Ag, or A9 and A10, or A10 and An may be optionally joined to form a cycloalkyl.
In another aspect of the present invention, there is provided a method, use or composition described above, or a ligand identified by a method described above, wherein the ligand has the structure of Formula II:
Figure imgf000009_0002
Formula II wherein Ei, E2, E3, E4, E5, E6, E9, E10 or En are independently selected from the group consisting of: H, an alkyl, a substituted alkyl, =0, =S, R-C=O, =N-R, NO2, CN, halogen, and SO2R where R is a 2 to 7 atom substituent; and at least one of Ei and E2 is selected from the group consisting of: =0, =S, R-C=O, =N-R, NO2, CN, halogens, and SO2R; E7 and Es are independently selected from the group consisting of: an alkyl, a substituted alkyl, -OH, -SH, -NH2, and -NHR, wherein at least one of E7 and E8 is selected from the group consisting of: -OH, -SH, -NH2, -NHR; and, E4 and E5, or E5 and E6 or E9 and E10, or E10 and Ei1 may be optionally joined to form a cycloalkyl group. In another aspect of the present invention, there is provided a method, use or composition described above, or a ligand identified by a method described above, wherein the ligand has the structure of Formula III:
Figure imgf000010_0001
Formula III wherein Bi, B2, B3, B4, B5, Bβ and B7 are independently selected from the group consisting of: H, an alkyl, a substituted alkyl, -OH, -SH, -NH2, and -NHR, =0, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, and ether-linked aliphatic rings having between 3 and 7 members and R is a 2 to 7 atom substituent, wherein at least one of B5, B^ or B7 is selected from the group consisting of: -OH, -SH, -NH2, and -NHR and at least one of Bi, B2 or B3 is selected from the group consisting of: =0, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, and ether-linked aliphatic rings having between 3 and 7 members; and B5 and B6 or B6 and B7 may be optionally joined to form a cycloalkyl.
In another aspect of the present invention, there is provided a method, use or composition described above, or a ligand identified by a method described above, wherein the ligand has the structure of Formula IV:
Figure imgf000011_0001
Formula IV wherein Di and D2, D3 and D4 are independently selected from the group consisting of =O, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, ether-linked aliphatic rings having between 3 and 7 members, -OH, -SH, -NH2, and -NHR, where R is a 2 to 7 atom substituent; D5, D6, D7, D§, D9 and D10 are independently selected from the group consisting of H, an alkyl and a substituted alkyl; and D5 and D6, or D6 and D7, or D7 and Ds, or Dg and D9, or D9 and D 10 may be optionally joined to form a cycloalkyl structure.
In another aspect of the present invention, there is provided a method, use or composition described above, or a ligand identified by a method described above, wherein the ligand has the structure of Formula V:
Figure imgf000011_0002
Formula V wherein Fi, F2, F3. F4, F5, Fβ, F7, Fg and F9 are independently selected from the group consisting of H, an alkyl, a substituted alkyl, -OH, -SH, -NH2, -NHR, =O, =S, R-C=O, =N-R, NO2, CN, halogen and SO2R, wherein at least one of Fi and F2 is selected from the group consisting of: -OH, -SH, -NH2, and -NHR, and at least one of F3 and F4 is selected from the group consisting of =0, =S, R-C=O, =N-R, NO2, CN, halogen and SO2R, where R is a 2 to 7 atom substituent.
In another aspect of the present invention, there is provided methods, uses and compositions described above, wherein the compound is a steroid.
In another aspect of the present invention, there is provided a use of a compound of this invention for treating a disease selected from the group consisting of: osteoporosis, female sexual dysfunction, endometriosis, anorexia nervosa, androgen producing ovarian cancer hyperthyroidism, hypogonadism (males), androgen insensitivity deficiency, alcoholic hepatic cirrhosis (males), and primary biliary cirrhosis (females), Klinefelter' s syndrome, teratogenic disorders, cervical dysplasia, ovarian dysfunction, male or female infertility, sexual or erectile dysfunction, decreased libido, vaginal dryness, thinning of the vaginal wall, menopause symptoms including hot flashes, osteopenia, diabetes, hyperglycemia, hyperglyceridemia, hypercholesterolemia, hypertension, atherosclerosis, cardiovascular disorders and disease, obesity, neurological dysfunction, Alzheimer's disease, dementia and cataracts. In some embodiments, the compound is not the compound is not a compound set out in Table 13 or 14. In some embodiments, the compound is not a steroid. The use may be for preparation of a medicament for treating the aforementioned disease.
In another aspect of the present invention, there is provided a method or use described above wherein the sex hormone is testosterone or estrogen.
In another aspect of the present invention, there is provided a method or use described above wherein the compound is selected from the group consisting of: l,l'-Spirobi[indane-5,6-diol], 3,3,3',3'-tetramethyl- (696),
4-[5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]ρhenol (2593), 4-[5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]-2-methoxy-phenol (2623), [3-(2-hydroxypropyl)-4-methyl-2-oxo-chromen-7-yl] acetate (6767), 3-(7-hydroxy-4,8-dimethyl-2-oxo-chromen-3-yl)propanoic acid (8590), l-(4-hydroxyphenyl)-3-pyridin-3-yl-prop-2-en-l-one (5636), and 1 -(2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)ethanone (5597).
In another aspect of the present invention, there is provided a computer implemented method for identifying a candidate ligand for sex hormone binding globulin (SHBG) comprising: I) providing a data structure comprising data for a plurality of chemical structures identifying a functionality for individual sites within each chemical structure according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; II) providing a data structure comprising data for a pharmacophore identifying a functionality for individual sites within the pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and spatial co-ordinates for each site having said functionality; and III) comparing the data structures of (I) and (II) and identifying a chemical structure in (I) matching the pharmacophore of (II); wherein the pharmacophore is: pharmacophore A, B, or C
In another aspect of the present invention, there is provided a computer implemented method described above, further comprising: IV) providing a data structure comprising data for the candidate ligand comprising said functionality and co-ordinates for sites within the candidate ligand; V) providing a data structure comprising data for a sex hormone binding site of SHBG including functionality of sites according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic and spatial co-ordinates for each site having said functionality; and VI) comparing the data structures of (IV) and (V) to provide a model showing presence or absence of binding of the ligand to the binding site.
In another aspect of the present invention, there is provided a computer implemented method described above, wherein the data structures (IV) and (V) further comprise a functionality for sites having spatial co-ordinates defining the position of a positive charge.
In another aspect of the present invention, there is provided a computer implemented method described above, wherein data structures (IV) and (V) further comprise co-ordinates defining size and shape of said ligand and said binding site respectively, wherein said model shows presence or absence of fit of said ligand with said binding site.
In another aspect of the present invention, there is provided a computer implemented method described above, further comprising: further comprising: VII) obtaining a compound comprising the chemical structure of candidate ligand; VIII) combining the compound with a protein containing the sex hormone binding site; and IX) detecting binding of the compound to the binding site.
In another aspect of the present invention, there is provided a computer implemented method described above, further comprising: further comprising measuring binding affinity of the compound for the binding site.
In another aspect of the present invention, there is provided a data structure embodied in a computer readable medium comprising means for storing a functionality for individual sites within a pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and means for storing spatial co-ordinates for each site having said functionality; wherein the pharmacophore is pharmacophore A, B or C.
In another aspect of the present invention, there is provided a data structure embodied in a computer readable medium comprising fields storing a functionality for individual sites within a pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and at least one field for storing spatial co-ordinates for each site having said functionality, wherein the pharmacophore is pharmacophore A, B, or C.
In some embodiments the compounds and ligands of the present invention are not the specific steroids described in Tables 13 or the compounds or substances described in Table 14.
Table 13 - List of steroid compounds with known binding affinities for SHBG
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Table 14 - List of non-steroid substance with known binding affinities for SHBG
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
In some embodiments the compounds and ligands of the present invention are not any steroid, including those listed in Table 14. The term "steroid" as used herein describes a group of compounds which have four fused rings and contain 17 carbon atoms in the fused ring structure. Steroids often have Sterane (1,2-cyclopentanoperhydrophenanthrene) as a core structure, wherein the core structure may be partially or complete hydrogenated and may contain functional substituent groups. Common substituents on the fused ring structure include a C1O methyl, a Q3 methyl as well as a C17 functional group such as a ketone, a hydroxyl or an alkyl.
A group of steroid hormones of particular interest are the sex hormones. These are typically produced by the adrenal cortex and/or the gonads. These hormones function generally to promote development and maintenance of the secondary sex characteristics and structures, prepare the female for pregnancy, and aid in development of gametes.
Sex hormones are further subdivided into three main categories: estrogens, progestagens and androgens. Estrogens are C^ steroids that always have an oxygenated substituent at C17, typically have a phenolic ring in the sterane structure with the hydroxy substituent at C3, and typically do not have a C1O substituent. 17β-estradiol is a typical example of an estrogen. Progestagens are C21 steroids with a en-4-one-3 substituent and a ketone substituent. Progesterone is a typical example of a progestagen. Androgens are Q9 steroids. Testosterone is a typical example of an androgen.
The term "alkyl" as used herein refers to straight or branched chain unsubstituted hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms.
The term "substituted alkyl" as used herein refers to an alkyl group as defined above having one, two, three, or four substituents selected from the group consisting of halogen, trifluoromethyl, alkenyl, alkynyl, nitro, cyano, keto (C=O), ORa, SRa, NRaRb, NRa SO2, NRa SO2 Rc, SO2 Rc, SO2 NRa Rb, CO2 Ra, C(=O)Ra, C(=O)NRa Rb, OC(=O)Ra, -OC(=O)NRa Rb, NRa C(=O)Rb, NRa CO2 Rb, =N-0H, =.N-O-alkyl, aryl, heteroaryl, heterocyclo and cycloalkyl, wherein Ra and Rb are selected from hydrogen, alkyl, alkenyl, cycloalkyl, heterocyclo, aryl, and heteroaryl, and Rc is selected from hydrogen, alkyl, cycloalkyl, heterocyclo aryl and heteroaryl. When a substituted alkyl includes an aryl, heterocyclo, heteroaryl, or cycloalkyl substituent, said ringed systems are as defined below and thus may in turn have zero to four substituents (preferably 0-2 substituents), also as defined below. When either Ra, Rb or Rc is an alkyl or alkenyl, said alkyl or alkenyl may optionally be substituted with 1-2 of halogen, trifluoromethyl, nitro, cyano, keto (=0), OH, O(alkyl), phenyloxy, benzyloxy, SH, S(alkyl), NH2, NH(alkyl), N(alkyl)2, NHSO2, NHSO2(alkyl), SO2(alkyl), SO2 NH2, SO2 NH(alkyl), CO2 H, CO (alkyl), C(=0)H, C(=O)alkyl, C(=0)NH2, C(=O)NH(alkyl), C(=O)N(alkyl)2, OC(=O)alkyl, -OC(=O)NH2, -OC(=O)NH(alkyl), NHC(=O)alkyl, and/or NHCO2 (alkyl).
The term "cycloalkyl" refers to fully saturated and partially unsaturated hydrocarbon rings of 3 to 9, preferably 3 to 7 carbon atoms. The term "cycloalkyl" includes such rings having zero to four substituents (preferably 0-2 substituents), selected from the group consisting of halogen, alkyl, substituted alkyl (e.g., trifluoromethyl), alkenyl, substituted alkenyl, alkynyl, nitro, cyano, keto, O-Ra, S-Ra, NRa-Rb, NRa, SO2, N-Ra SO2, Ra, C(=0)H, acyl, -CO2-H, alkoxycarbonyl, carbamyl, sulfonyl, sulfonamide, -OC(=O)-Rb, =N-OH, =N-O-alkyl, aryl, heteroaryl, heterocyclo, a 4 to 7 membered carbocyclic, wherein Ra and Rb are defined as above. The term "cycloalkyl" also includes such rings having a phenyl ring fused thereto or having a carbon-carbon bridge of 3 to 4 carbon atoms. Additionally, when a cycloalkyl is substituted with a further ring, i.e., aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclo, heterocycloalkyl, cycloalkylalkyl, or a further cycloalkyl ring, such ring in turn may be substituted with one to two of C(o-4) alkyl optionally substituted with halogen, trifluoromethyl, alkenyl, alkynyl, nitro, cyano, keto (=O), OH, O(alkyl), phenyloxy, benzyloxy, SH, S(alkyl), NH2, NH(alkyl), N(alkyl)2, NHSO2, NHSO2(alkyl), SO2(alkyl), SO2 NH2, SO2 NH(alkyl), CO2H, CO2 (alkyl), C(=O)H, C(=O)alkyl, C(=0)NH2, C(=O)NH(alkyl), C(=O)N(alkyl)2, OC(=O)alkyl, -OC(O)NH2, -OC(O)NH(alkyl), NHC(O)alkyl, and NHCO2 (alkyl).
Brief Description of the Drawings:
In drawings which illustrate embodiments of the invention,
Figure 1 a) shows graphical depictions of a representative pharmacophore A superimposed with the structure of 5α-dihydrotestosterone (DHT). b) shows graphical depictions of a representative pharmacophore B superimposed with the structure of DHT. c) shows graphical depictions of a representative pharmacophore C superimposed with the structure of DHT.
Figure 2 a) illustrates the distances between the individual features of an embodiment of pharmacophore A. b) illustrates the angles between the individual features of the embodiment in 2(a). c) illustrates the distances between the individual features of an embodiment of pharmacophore B. d) illustrates the angles between the individual features of the embodiment in 2(c). e) illustrates the distances between the individual features of an embodiment of pharmacophore C. f) illustrates the angles between the individual features of the embodiment in 2(e).
Figure 3 shows the configuration of the Artificial Neural Network (ANN) containing 28 input, 8 hidden and 1 output nodes, used for ranking SHBG ligands.
Figure 4 depicts the structure of a portion of human SHBG containing DHT (a) or estradiol (b) in the steroid-binding pocket.
Figure 5 shows the relationship plot between experimental association constant (LogKa) and the consensus docking score.
Figure 6 shows a superimposition of DHT molecules docked into SHBG using Glide software (light) with bound DHT configuration defined from the crystal structures (dark).
Figure 7 shows the distribution of the docking consensus score for pharmacophore-identified compounds and randomly derived molecular structures.
Figure 8 shows the displacement curves for test compounds used in the competition assay to determine the relative binding affinities of human SHBG ligands. The amount of [3H] DHT bound to SHBG in the presence of increasing concentrations of competitor ligands (B) is expressed as a percentage of the amount of [3H] DHT bound to SHBG in the absence of competitor ligand (Bo).
Figure 9 shows the structural alignment of a compound l-(2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)ethanone (5597) with other SHBG ligands. a) Structure of non-steroidal SHBG ligand 5597, identified herein, (light scaffold) flexibly aligned with (-)-3, 4-divanyllyltetrahydrofuran, a known non-steroidal SHBG ligand (dark scaffold), b) Structure of 5597 in its SHBG-docked configuration superimposed with the experimentally determined orientation of DHT in the SHBG binding site c) Structure of 5597 flexibly aligned with a dicyclohexyl derivative previously demonstrated to have binding affinity toward the rat ABP protein [14]. Detailed Description of the Invention:
These common features and relationships between these features of SHBG binding ligands is described by the following pharmacophores:
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000026_0002
Figure imgf000027_0001
acophore A: Range of possible (x,y,z) co-ordinates for each feature
Feature X y Z
Fl 27.8-25.8 13.3-11.3 31.4-29.4
F2 25.2-23.2 11.7-9.7 33.3-31.3
F3 20.1-18.1 10.7-8.7 34.9-32.9
F4 29.2-27.2 12.9-10.9 29.9-27.9
F5 28.8-26.8 15.1-13.1 31.2-29.2
F6 17.9-15.9 9.8-7.8 34.2-32.2
F7 15.8-13.8 9.5-7.5 34.2-32.2
F8 17.9-15.9 7.9-5.9 33.3-31.3
F9 17.7-15.7 8.5-6.5 35.9-33.9
Pharmacophore B: Range of possible (x,y,z) co-ordinates for each feature
Feature X y Z
Fl 24.7-22.7 9.6-7.6 38.3-36.3
F2 21.0-19.0 13.5-11.5 28.9-26.9
F3 21.8-19.8 14.7-12.7 31.6-29.6
F4 21.0-19.0 13.8-11.8 26.9-24.9
F5 22.3-20.3 12.7-10.7 31.9-29.9
F6 23.7-21.7 11.3-9.3 35.7-33.7
F7 19.0-17.0 13.3-11.3 28.5-26.5
Pharmacophore C: Range of possible (x,y,z) co-ordinates for each feature
Feature X y Z
Fl 2.5-0.5 6.6-4.6 1.9-0.1
F2 1.7-0.3 9.0-7.0 3.2-1.2
F3 2.3-0.3 4.6-2.6 1.6-0.4
F4 0.8-1.2 12.3-10.3 4.1-2.1
F5 4.1-2.1 6.3-4.3 0.9-1.1
F6 1.1-0.9 17.1-15.1 4.7-2.7
F7 1.0-1.0 14.8-12.8 3.7-1.7
F8 1.7-0.3 18.9-16.9 4.4-2.4
F9 0.6-1.4 18.5-16.5 6.2-4.2 The features listed in the Tables above are often dependent on the relative positioning of electrons. As a result, it is convenient to describe these features as sites having centers in a pharmacophore. Sites are areas of a pharmacophore that define regions where the desired structural features will exist in a compound, relative to each other. Spatial positions (x,y,z) of sites are described with respect to their centers and the distance in angstroms between two sites is measured with respect to their centers.
Hydrogen bond acceptor/donor, acceptor and donor features are features that are capable of: i) accepting a hydrogen bond from hydrogen bond donor, ii) donating a hydrogen bond to a hydrogen bond acceptor or iii) capable of both. A hydrogen bond is formed by three atoms: one hydrogen atom and two electronegative atoms. The hydrogen atom is covalently bonded to one of the electronegative atoms (the hydrogen bond donor), the other electronegative atom is the hydrogen bond acceptor. The two electronegative atoms take up some electron density from the hydrogen atom, and as a result, each electronegative atom carries partial negative charge and the hydrogen atom carries partial positive charge. The hydrogen atom and the hydrogen bond acceptor can then have attractive interactions. For a review of hydrogen bonding, see [19].
Hydrogen bond acceptors include =0, =S, R-C=O, =N-R, NO2, CN, halogens, SO2R, ether-linked aliphatic rings of between 3 and 7 members. Hydrogen bond donors include -OH, -SH, -NH2, -NHR, where R is a 2 to 7 atom substituent. Polar group substituents include =S, SO2R, -OH, =NR, =0, -CN, halogens, RCOOH, COH, NO2. Halogens include chlorine, bromine, fluorine and iodine.
Hydrophobic/aromatic sites are features that have aromatic structures or hydrophobic structures or both. Aromatic structures are typically planar, cyclic structures where all of the atoms in the ring or rings are sp2 and the structure has a free "p" orbital in which Pi electrons can travel through so as to be distributed throughout the structure. Aromatic structures are typically resonance stabilised and have 4n + 2Pi electrons, wherein n is any integer. Benzene rings, naphthyl rings, benzene rings substituted with a heteroatom, and naphthyl rings substituted with a heteroatom are typical examples of aromatic structures. Hydrophobic structures are non-polar structures that do not readily interact with or dissolve in water. Typical examples of hydrophobic structures include unsubstituted alkanes, alkyls, and alkenyls. Often aromatic structures are also hydrophobic structures.
Sites of substitution unrestrained by the pharmacophore may be found on aromatic or aliphatic or heteroorganic ring structures and double bond structures of the parent molecule, as well as other sites. Many of these sites are not as restricted by the pharmacophores as are sites of hydrogen bond donor/acceptors.
A projection point is a feature on a pharmacophore that restricts or defines the outer extent of a hydrogen bond donor/acceptors site, a hydrogen bond donor site or a hydrogen bond acceptor site in one direction from the centre. A plurality of projection points provides information concerning both shape and size of the particular a hydrogen bond donor/acceptor site, a hydrogen bond donor site or a hydrogen bond acceptor site to which the projection point is associated.
The pharmacophores described herein can be used to screen electronic libraries of compounds using techniques known to one skilled in the art (see, for example, Y. C. Martin, 3D Database Searching in Drug Design, J Med. Chem. 35, 2145(1992); A. C. Good and J. S. Mason, Three Dimensional Structure Satabase searches, Reviews in Comp. Chem. 7, 67 (1996); Guner, O. F. Pharmacophore perception, Development, and Use in Drug Design, IUL Biotechnology Series; International University Line: La Jolla, CA, (2000); Klebe, G. Virtual Screening: An Alternative of Complement to High Throughput Screening?, Kluwer: Dordrecht, The Neherlands, (2000); and Virtual Screening for Bioactive Molecules, Bohm, H. J., Schneider, G., Kubinyi, H., Manhold, R., Timmerman, H. Eds., Methods and Principles in Medicinal Chemistry, Wiley: New York, (2000)).
In order for a library to be able to be screened using a pharmacophore, the relevant structural information must be available. Structural data on a panel of compounds may be selected from multiple sources, including combinatorial libraries, compound collections and other sources. The structural data is most frequently presented in a database format. Examples of suitable databases include Dictionary of Natural Products DNP (www.crcpress.com), Bioactive Natural Products (Szenzor Co., Hungary), BioScreenNP files (www.ibscreen.com/natural.shtml), Marine Natural Product Database MNPD [Lei, J., Zhou, J. A Marine Natural Product Database. /. Chem. Inf. Comp. ScI 42: 742-748 (2002)], BioSPECS natural products database (BioSPECS Inc., the Netherlands), ChemDiv natural products database (ChemDiv Inc., San Diego, CA), InterBioScreen database (InterBioScreen Ltd., Russia), Herbal medicine index (www.micromedex.com), Traditional Chinese Medicine Database [He, M., Yan, X., Zhou, J., Xie, G. Traditional Chinese Medicine Database and Application on the Web. /. Chem. Inf. Comp. Sci. 41: 273-277 (2001)], 3D Database of Components from Chinese Traditional Medicinal Herbs [Qiao, X., Hou, T., Zhang, W., Guo, S., Xu, X. A 3D Structure Database of Components from Chinese Traditional Medicinal Herbs J. Chem. Inf. Comp. Sci, 42: 481-489 (2002)], Assinex collection (www.assinex.com), Maybridge catalogue (www.maybridge.com), the Merck index (http://chemfinder.cambridgesoft.com/reference/TheMerckIndex.asp).
Compounds identified by screening of electronic libraries using pharmacophores can be prioritised using further techniques, including quantitative structure-activity relationship (QSAR) techniques, artificial neural networks (ANN) and virtual docking techniques. There are many ways to apply these techniques and it is within the understanding of one skilled in the art to apply these techniques appropriately in order to prioritise the hits obtained by screening an electronic library using a particular pharmacophore. (See, for example, Cherkasov, A. 'Inductive' Descriptors. 10 Successful Years in QSAR. Curr Comp Aided Drug Design. 2005, 1, 21-42; Cherkasov, A. Inductive QSAR Descriptors. Distinguishing Compounds with Antibacterial Activity by Artificial Neural Networks. Intern J MoI Sci. 2005, 6, 63-86; and Cherkasov, A., Jankovic, B. Application of 'Inductive' QSAR Descriptors for Quantification of Antibacterial Activity of Cationic Polypeptides. Molecules, 2004, 9, 1034-1052). Details of exemplary techniques that may be applied are discussed in more detail in Examples 1, 2 and 3. For in silico discovery of non-steroidal SHBG ligands both structure-based and ligand-based techniques may be employed, as well as QSAR (quantitative structure-activity relationships) solutions. Structure libraries suitable for this purpose may be combinatorial, or natural product libraries, and may be of varying size. Larger libraries allow for greater numbers of compounds to be considered. Natural compound databases are typically rich in biologically active substances.
Each of the pharmacophores described herein are useful for identifying different types of SHBG binding ligands. In particular, pharmacophore A is preferably for identifying steroidal ligands, but will often identify less ligands than Pharmacophores B and C. Pharmacophore C is preferable for identifying non-steroidal ligands and will often identify the largest number of ligands, and in rare cases, may also identify some false positive hits that will require wet assaying to identify as false positives. Pharmacophores B will identify steroidal ligands and non-steroidal ligands, and often identify more ligands than pharmacophore A, but less than pharmacophore C. Each of pharmacophore A, B and C are capable of identifying ligands that the other pharmacophores would not identify. Using combinations of the pharmacophores will identify ligands that have the properties associated with each of the pharmacophores used. Ligands identified using combinations of the pharmacophores will likely have features common to steroidal ligands and non-steroidal ligands. Identification of SHBG ligands with no intrinsic biological properties of their own (or with properties selected for a particular effect) which block or compete for sex hormone binding represent a means of altering the role of SHBG in the body. For example, use of such ligands which have no intrinsic steroidal activity may enhance the bioavailability and activity of endogenous sex hormones.
Known ligands of SHBG [18] were used to develop pharmacophore models of the SHBG steroid-binding site. These pharmacophores were then used to screen an electronic library of compounds. This led to identification of a smaller set of structures that met the stringent structural requirements imposed by these pharmacophores. The known set of SHBG ligands were then used to train a QSAR model algorithm to differentiate known SHBG ligands from other structures. The resulting structure-activity model was then applied to rank the pharmacophore-identified compounds for their potential binding affinity toward SHBG. Virtual docking studies of selected structures in the SHBG steroid-binding pocket were conducted, and structures that ranked highly by both the QSAR model and/or were favoured by the virtual docking, were subsequently subjected to experimental testing of their potencies as SHBG ligands, using wet assays.
This computational approach resulted in the identification of 105 prospective compounds from a collection of 23,836 natural substances that were prioritised for testing by QSAR modelling and virtual docking techniques. This procedure resulted in the identification of eight novel non-steroidal ligands with an ability to displace the natural ligand (DHT) with the highest known affinity for human SHBG at low micro-molar concentrations. The QSAR model assigned very significant network outputs above 0.5 to four of the most active compounds which produce a >35% reduction of [3H] DHT binding to SHBG in the screening assay (compounds l,l'-Spirobi[indane-5,6-diol], 3,3,3',3'-tetramethyl- (696), 4-[5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]phenol (2593), 4-[5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]-2-methoxy-phenol (2623), 4-[5-(hydroxymethyl)-2-methyl-7-oxabicyclo[3.3. l]non-2-en-8-yl]phenol (2228)). This model also resulted in output values above 0.10 for four other molecules ([3-(2-hydroxypropyl)-4-methyl-2-oxo-chromen-7-yl] acetate (6767), 3-(7-hydroxy-4,8-dimethyl-2-oxo-chromen-3-yl)propanoic acid (8590), l-(4-hydroxyphenyl)-3-pyridin-3-yl-prop-2-en-l-one (5636), l-(2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)ethanone (5597)) that were subsequently shown to bind to SHBG. Assays that may be used to test the binding affinity of a particular compound to SHBG are known by those of one of skill in the art. These assays include competition assays using rat prostate androgen receptor [16], competition assays using rat, rabbit, money or human epididymal androgen binding protein [16], competition assays using human sex hormone binding globulin, [16] screening assays based on the ammonium sulfate precipitation method [17], competitive binding assays in diluted human pregnancy serum [17], and charcoal assays using varying concentrations of the testing compound and tritiated DHT [12]. A detailed example of a particular binding assay that may be used is described in Example 4. Additional examples of assays that may be used to determine the binding affinity of a particular ligand to SHBG may be found in Jury, et al., Journal of Steriod Biochemistry & Molecular Biology, 75 (2000) 167-176; Winneker, et al, J. Med., Chem. 33 (1990), 129-132; Rousseau, G.G. et al, J Steroid Biochem. 31 (1988), 691-697; Rousseau, G.G., et al, Nature. 284 (1980), 458-459; and Schottner, M., etal., J Nat Products. 61 (1998), 119-121.
The binding affinities of selected testing candidates as determined by a competitive displacement assay described in Example 4 are shown in Figure 8. The affinity of compounds 2623 and 5597 exceed those of other synthetic and natural non-steroidal compounds with endocrine disrupting properties, which typically bind to SHBG steroid-binding site with association constants in the range of 0.02 x 105 M"1 to 8 x 105 M'1 [12, 13, 20].
Figure 9a shows a superimposition of the molecular structures of 5597 and (-)-3, 4-divanyllyltetrahydrofuran, a natural non-steroidal lignan with a high association constant for SHBG [12]. The overall shape of these molecules is similar, and critical chemical groups required for SHBG binding are positioned closely in space. The ability of compound 5597 and others to displace DHT and estradiol from SHBG indicates that these non-steroidal compounds identified interact with the SHBG steroid-binding pocket. Figure 9b shows the structure of 5597 docked into the active site of SHBG and superimposed against bound DHT. In this orientation, 5597 reproduces all critical binding features of a sex steroid.
Two other non-steroidal ligands of SHBG (compounds 2623 and 2593) are structurally very similar but differ in their binding affinities. The main structural difference between them is the presence of a methoxy-group adjacent to a hydroxyl group on a phenolic ring structure. A similar difference is also observed in the relative binding affinities of estradiol and 2-methoxy-estradiol for human SHBG [18], suggesting that the phenolic ring structures of compounds 2623 and 2593 reside within the SHBG steroid-binding site in the same orientation as ring A of an estrogen molecule. The compounds identified using techniques described herein are suitable for treating diseases that are characterised by undesirable levels, high or low, of sex hormone in the plasma. Examples of such diseases include, osteoporosis, female sexual dysfunction, endometriosis, anorexia nervosa, androgen producing ovarian cancer hyperthyroidism, hypogonadism (males), androgen insensitivity deficiency, alcoholic hepatic cirrhosis (males), and primary biliary cirrhosis (females), Klinefelter's syndrome, teratogenic disorders, cervical dysplasia, ovarian dysfunction, male or female infertility, sexual or erectile dysfunction, decreased libido, vaginal dryness, thinning of the vaginal wall, menopause symptoms including hot flashes, osteopenia, diabetes, hyperglycemia, hyperglyceridemia, hypercholesterolemia, hypertension, atherosclerosis, cardiovascular disorders and disease, obesity, neurological dysfunction, Alzheimer's disease, dementia and cataracts.
These diseases are treatable using compounds described herein because the ligands that have been identified using the pharmacophores described herein are capable of binding to sex hormone binding globulin (SHBG) and consequently competing with sex hormones for binding to SHBG. By competing with sex hormones, ligands identified herein can increase the concentration of available sex hormone in a mammal.
Ligands that bind to SHBG may also be coupled to or synthesised to include known labelling moieties such as radio-isotopes and then used for imaging. Such labelled compounds are useful for imaging certain types of cancer, particularly estrogen secreting cancers. In these types of cancer, the labelled ligand is sequestered at the site of the cancer because the estradiol secreted by the cancer displaces the labelled ligand more frequently at the cancer site. High concentration of estradiol at these cancer sites results in a higher concentration of labelled ligand being displaced at the cancer site. Examples of labelling ligands of SHBG can be found in Seimbille et al. Steroids 67, (2002) 765-775.
Alternatively, a desired moiety such as a cytotoxic compound, for example cisplatin or other anti-cancer agents could be coupled to a SHBG ligand. Such moieties are delivered and deposited at a desired site such as an estrogen secreting cancer. Cytotoxic moieties coupled to ligands in this way could be more effective as therapeutics by minimising side effects from the cytotoxic agent on the patient since more of the cytotoxic agent would be delivered to the desired site and less cytotoxic compound would be delivered to undesired sites. Examples of suitable target cancers include endometrial cancer, cervical cancer, uterine cancer and breast cancer. Labelling and coupling to SHBG ligands as described above could be applied to areas and tissues having a high androgen concentration, for example androgen secreting ovarian tumors, as well as areas and tissues with high estrogen concentrations.
Compounds that bind to SHBG can be described by the following formulas I, II, III, IV and V:
Figure imgf000035_0001
Formula I
Substituent group A1 functions generally as a hydrogen donor group. Hydrogen bond donor groups include -OH, -SH, -NH2, -NHR, where R is a 2 to 7 atom substituent. Substituent group A7 functions generally as a hydrogen acceptor group. Hydrogen bond acceptor groups include =0, =S, R-C=O, =N-R, NO2, CN, halogens, SO2R, ether-linked aliphatic rings of between 3 and 7 members. Substituents groups A2, A3, A4, A5, A$, As, A9, Aio, An or Ai2 may be alkyl groups or substituted alkyl groups. Substituent groups A3 and A4, or A4 and A5, or A6 and A7, or A7 and A§, or A9 and Aio, or Aio and An may be joined as cycloalkyl groups.
Synthesis of compound 5597 and derivatives depicted generally in Formula I may be prepared according to methods described in US 6677350, US 5208343, Kenkyu Hokuku - Sen'i Kobunshi Zairyo Kenkyusho 155:45-8 (1987), J. Appl. Polym. Sci 32:4127-35 (1986), Bulletin de Ia Societe Chimique de France (1961) 584-6, JP 58025303, JP54117482 and DE 1921348, and other methods known in the art.
Figure imgf000036_0001
Formula II
Substituent groups Ei or E2 function generally as hydrogen acceptor groups. Hydrogen bond acceptor groups include =0, =S, R-C=O, =N-R, NO2, CN, halogens or SO2R, where R is a 2 to 7 atom substituent. Substituent groups E7 or Es function generally as hydrogen donor groups. Hydrogen bond donor groups include -OH, -SH, -NH2, -NHR, where R is a 2 to 7 atom substituent. Substituent groups Ej, E2, E3, E4, E5, Eg, E7 Eg, E9, E10 or En may be alkyl groups or substituted alkyl groups. Substituent groups E4 and E5, or E5 and Eg or E9 and E10, or Eio and En may be joined as cycloalkyl groups.
Synthesis of compound 5636 and derivatives as depicted generally in Formula II may be prepared according to methods described in US 6677350, US 5208343, Kenkyu Hokuku - Sen'i Kobunshi Zairyo Kenkyusho 155:45-8 (1987), J. Appl. Polym. Sci 32:4127-35 (1986), Bulletin de Ia Societe Chimique de France (1961) 584-6, JP 58025303, JP54117482 and DE 1921348 and other methods known in the art.
Figure imgf000036_0002
Formula III
Substituent groups B5, Bg or B7 function generally as hydrogen donor groups. Hydrogen bond donor groups include -OH, -SH, -NH2, -NHR, where R is a 2 to 7 atom substituent. Substituent groups Bi, B2 or B3 functional generally as hydrogen acceptor groups. Hydrogen bond acceptor groups include =0, =S, R-C=O, =N-R, NO2, CN, halogens, SO2R, ether-linked aliphatic rings of between 3 and 7 members. Substituent groups Bi, B2, B3, B4, B5, B6 or B7 may be alkyl groups or substituted alkyl groups. Substituent groups B5 and Be or B6 and B7 may be joined as cycloalkyl groups.
Synthesis of compound 6767 or 8590 and derivatives depicted generally as Formula III may be prepared according to methods described in J. Appl. Microbiol. 91:1118-1130 (2001), Tx. J. Science 17:340-4 (1965), Khimiya Geterotsiklicheskikh Soedinenii 2:150-4 (1970) and GB 1044608, and other methods known in the art.
Figure imgf000037_0001
Formula IV
Substituent groups Di and D2, D3 and D4 function generally as hydrogen donor and hydrogen acceptor groups. Hydrogen bond acceptors include =O, =S, R-C=O, =N-R, NO2, CN, halogens, SO2R, ether-linked aliphatic rings of between 3 and 7 members. Hydrogen bond donors include -OH, -SH, -NH2, -NHR, where R is a 2 to 7 atom substituent. Substituent groups D5, Dg, D7, Dg, D9 and Djo may be alkyl groups or substituted alkyl groups. Substituent groups D5 and Dβ, or Dg and D7, or D7 and Dg, or Dg and D9, or D9 and D10 may be joined as cycloalkyl structures.
Synthesis of compound 696 and derivatives depicted generally as Formula IV may be prepared according to methods described in JP 09110771, Polym. Mater. Sci. Eng. 70:378-9 (1993), JP 03148232, JP 02286642, ip 03386641, JP 02248954, EP 342035, EP 307951, Bur. Polym. J. 15(7):631-8 (1979), FR 2322161, Izv. Akad, Nauk. SSSR. Ser. Khim- (12):2808-10 (1973) and Tetrahedron Lett. (34):3707:10 (1968), and other methods known in the art.
Figure imgf000038_0001
Formula V
Substituent groups Fi or F2 function generally as hydrogen donor groups. Hydrogen bond donor groups include -OH, -SH, -NH2, -NHR, where R is a 2 to 7 atom substituent. Substituent groups F3 or F4 function generally as hydrogen bond acceptor groups. Hydrogen bond acceptor groups include =O, =S, R-C=O, =N-R, NO2, CN, halogens or SO2R, where R is a 2 to 7 atom substituent. Substituent groups Fi, F2, F3, F4, F5, F6, F7, F$ or F9 may be alkyl groups or substituted alkyl groups.
Compounds 2593, 2623 and derivatives depicted generally in Formula V may be synthesised and modified according to methods known in the art.
Compounds 696, 2593, 2623, 6767, 8590, 5636, 5597 and derivatives are also available from several chemical library suppliers, including Vitas-M Labs, ChemBridge, Ambinter, Asinex Laboratories, Interchim Intermediates or InterBioScreen, Ltd.
The efficacy of the compounds described herein may be measured by performing an SHBG binding assay to determine the binding affinity of a particular ligand. Depending on the disease being treated a particular ligand can be selected based on its binding affinity for SHBG. For example, if the disease is characterized by low levels of bio-available sex hormone, such as female sexual dysfunction or osteoporosis, then a ligand with a high binding affinity for SHBG will be chosen so that it occupies a ligand binding pocket of SHBG for longer periods, thereby preventing the bio-available androgens from binding to SHBG and encouraging more bio-available androgen hi the plasma.
Alternatively, if the ligand is to be used for imaging purposes, then a lower binding affinity for SHBG may be desired so that it becomes easier for the imaging agent to be displaced by a sex hormone. For example, a ligand to be labeled may be selected so that is has an affinity that is moderately or slightly weaker than a sex hormone that is produced by the target cancer tissue. Such a labeled ligand will stay bound to SHBG with a maximum efficiency until it contacts a sex hormone that has a higher binding affinity for SHBG. This is mostly likely to occur at the cancer site and displacement of the ligand will result in deposition of the imaging agent in the cancer site, thereby providing a better image.
Alternatively, if the ligand is coupled to a cytotoxic moiety, then a lower binding affinity for SHBG may be desired so that it becomes easier for the cytotoxic moiety to be displaced by a sex hormone, for example a sex hormone being produced by the target cancer. For example, a ligand to be coupled may be selected so that is has an affinity that is moderately or slightly weaker than a sex hormone that is produced by the target cancer tissue. Such a coupled ligand will stay bound to SHBG with a maximum efficiency until it contacts a sex hormone that has a higher binding affinity for SHBG. This is mostly likely to occur at the cancer site and displacement of the ligand will result in deposition of the cytotoxic moiety in the cancer site, thereby providing improved therapy with less side effects.
Compounds of this invention or for use in this invention may be water soluble and may be formed as salts. In such cases, pharmaceutical compositions in accordance with this invention may comprise a salt of such a compound, preferably a physiologically acceptable salt, which are known in the art. Pharmaceutical preparations will typically comprise one or more carriers acceptable for the mode of administration of the preparation, be it by injection, inhalation, topical administration, lavage, or other modes suitable for the selected treatment. Suitable carriers are those known in the art for use in such modes of administration.
Suitable pharmaceutical compositions may be formulated by means known in the art and their mode of administration and dose determined by the skilled practitioner. For parenteral administration, a compound may be dissolved in sterile water or saline or a pharmaceutically acceptable vehicle used for administration of non- water soluble compounds. For enteral administration, the compound may be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule may be enteric coated, or in a formulation for sustained release. Many suitable formulations are known, including, polymeric or protein microparticles encapsulating a compound to be released, ointments, gels, hydrogels, or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant may be employed to provide release over a prolonged period of time. Many techniques known to skilled practitioners are described in Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20th ed., Williams & Wilkins, (2000). Formulations for parenteral administration may, for example, contain excipients, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
EXAMPLE 1
Flexible Alignment and Pharmacophore Generation, Pharmacopohre based Virtual Screening or Novel SHBG Ligands, and Identification of 'drug-like' data subset
Pharmacophore A was developed from the structures of 5-alpha-dihydrotestosterone (DHT) and its close derivatives (Figure Ia, 2a, 2b). This pharmacophore model included nine major features (Table 4): two hydrophobic/aromatic sites (F2, F3), one hydrogen bond donor/acceptor point (F6) with three donor/acceptor projection points (F7, F8 and F9) and one hydrogen bond acceptor (Fl) with two acceptor projection points (F4 and F5).
Table 4 Pharmacophore A coordinates
Figure imgf000040_0001
Pharmacophore B was generated by aligning the crystallographic configurations of DHT and estradiol inside the steroid-binding pocket of SHBG, and it utilised seven basic features (Table 5): three hydrophobic/aromatic centers (F3, F5, F6), one H acceptor (Fl) and one H-bond donor (F2), with two donor projection points (F4, F7) (Figure Ib, 2c, 2d).
Table 5 Pharmacophore B co-ordinates
Figure imgf000042_0001
Pharmacophore C relied on the flexible alignment of a large number of known ligands of human SHBG [18], which resulted in the identification of the following common features (Table 6): three hydrophobic/aromatic centers, one hydrogen donor site with two donor projection points and one H-bond acceptor with two projection points (Figure Ic, 2e, 2f).
Table 6 Pharmacophore C co-ordinates
Figure imgf000042_0002
To test the adequacy of the developed pharmacophores, they were applied to the 16 strongest SHBG binders (compounds with >35% displacement of [3H] DHT from SHBG in the screening assay including entries 1, 2, 4, 5, 7-10, 12-15, 17-19 and 21 from Table 8); the 16 moderately strong SHBG binders (i.e., those that displaced 10-35% [3H] DHT from SHBG in the screening assay; namely, entries 3, 6, 11, 16, 20, 28, 29, 32, 33, 38, 41, 44, 46, 51-53) and 70 non-binders (entries 82-151). The pharmacophore performance was assessed through the predictions by the true hit-rate, false hit-rate and recovery-rate. The corresponding parameters for the three pharmacophores are presented in Table 7.
These pharmacophore models were then used as data-mining instruments of varying stringency. By applying them in different combinations to conformer sets generated for the 23,836 'drug-like' natural substances, 201 initial hits were identified, including 96 known steroid-like and 105 non-steroidal compounds (entries 283-388 of Table 8).
Table 7. Test results for pharmacophore hypotheses.
Total NonTrue hit Recovery False hit
Model Feature Steroids Unknown Inactive hits steroids rate rate rate
A 9 9 9 0 0 0 1 0.56 0
B 7 12 10 1 1 0 0.92 0.69 0
C 9 22 11 4 1 6 0.68 0.94 0.27
True hit rate = number of true hits / total number of hits False hit rate = number of false hits / total number of hits Recovery rate = number of true hits / total number of true hits
Table 8: Compounds used for in silico screening
Figure imgf000043_0001
Compounds with high binding affinity to SHBG
O=C1C=C2CC[C@ @H]([C@H](CC[C@H](O)[C@@
1 Testosterone 1.0 ](CXCC[C@@H]3([C@@](C)(CC1)2))1)1)3
O=C1CC[C@ @](C)([C@ @H](CC[C@ @H]([C@H](C
2 DHT 1.0 C[C@H](O)tC@ @](C)(CC[C@@H]23)4)4)2)Cl)3
Oclccc2c(cl)CC[C@@H]([C@H](CC[C@H](O)[C@
3 Estradiol 1.0 @](C)(CC[C@H]21)2)2)1 ANN
# Compound SMILES output
O=C1C[C@H](C)[C@@](C)([C@@H](CC[C@ @H]([
4 lα-Methyl-DHT 1.0 C@H](CC[C@H](O)[C@@](C)(CC[C@@H]23)4)4)2 )C1)3
O=C1CC[C@@](C)([C@H](C1)C[C@ @H](C)[C@@
5 7α -Methyl-DHT 1.0 H]([C@H](CC[C@H](O)[C@@](C)(CC[C@ @H]12)3 )3)1)2
O=C 1 CC[C @ @](C)([C@ @H](CC[C@ @H]([C@H](C
6 17-Methyl-DHT 1.0 C[C@@](O)(C)[C@@](C)(CC[C@@H]23)4)4)2)C1) 3
O=C1CC[C@@](C)([C@HJ(C1)C[C@ @H](C)[C@ @
7 7α ,17-Dimethyl-DHT 1.0 H]([C@H](CC[C@ @](O)(C)[C@@](C)(CC[C@@H] 12)3)3)1)2
F[C@@H](C[C@@H]([C@H](CC[C@H](O)[C@@](
8 6α -Fluoro-DHT 1.0 C)(CC[C@@H]1([C@@](C)(CCC(=O)C[C@H]23)3)) 3)3)1)2
5-Androstene-3β, O[C@ @H](CC[C@ @](C)(C(=CC[C@ @H]([C@H](C
9 1.0 17β-diol C[C@H](O)[C@@](C)(CC[C@ @H]12)3)3)1)C1)2)1
5-Androstane-3β, O[C@@H](CC[C@@](C)(C(=CC[C@@H]([C@H](C
10 1.0 17β-diol C[C@H](O)[C@@](C)(CC[C@@H]12)3)3)1)C1)2)1
7α -Methyl-5α
O=C1CC[C@@](C)(C(CC[C@@H]([C@H](CC[C@
11 -androstane-3βb, 17 β-dio 1.0 H](O)[C@ @](C)(CC[C@ @H]23)4)4)2)=C1C)3 1
O=C1C=C2C[C@ @H](C)[C@ @H](C3=CC[C@H](O)
12 4-Methyl-testosterone 1.0 [C@@](C)(CC[C@H]([C@@H](CC1)2)1)3)1
7α
O[C@ @H](CC[C@ @H](C(=CC[C@ @H]([C@H](CC
13 -Methyl-14-dehydro- 19- 1.0 [C@H](O)[C@@](C)(CC[C@H]12)3)3)2)C2)1)2 nortestosterone
5- Androstene- 19-nor-3 β O[C@H](C=C1CC[C@ @H]([C@H](CC[C@H](O)[C
14 1.0 ,17β-diol @ @](C)(CC[C@@H]2(tC@@](C)(CC3)l))l)l)2)3
O[C@ @H](CC[C@ @](C)(C(=CC[C@ @H]([C@H](C
4-Androstene-3β,17β-di
15 1.0 C[C@ @](O)(C#C)[C@ @](C)(CC[C@ @H]12)3)3)1)C ol 1)2)1 ANN
# Compound SMILES output
Oclccc2c(ccc3c2CC[C@ @](C)([C@ @H](CC[C@H](
16 17-Ethinyl-delta-5AD 1.0 O)2)3)2)cl
Oclcc2CC[C@ @H]([C@H](CC[C@H](O)[C@ @](C)
17 Hihydroequilenin- 17β 1.0 (CC[C@H](c2cclOC)l)2)2)l
O[C@@H](CC[C@ @H](C(=CC[C@ @H]([C@H](CC
18 2-Methoxyestradiol 1.0 [C@H](O)[C@ @](C)(CC[C@H]12)3)3)2)C2)1)2
O=ClC=CfCO @](C)(C(=C1)CC[C@ @H]([C@H](C
19 5-Estrene-3 β,17β-diol 1.0 C[C@ @](O)(C)[C@@](C)(CC[C@ @H] 12)3)3)1)2
O=C1CC[C@ @](C)([C@ @H](CC[C@ @H]([C@H](C
17-Methyl-l-dehyrdotes
20 1.0 C[C@ @](O)(C#C)[C@ @](C)(CC[C@ @H]23)4)4)2)C tosterone 1)3
O=C1C=C2CC[C@ @H]([C@H](CC[C@ @](O)(C#C)
21 17-Ethinyl-DHT 1.0 [C@](CC[C@H]([C@@H](CC1)2)1XCC)2)2)1
O=C1C=C2CC[C@ @H]([C@H](CC[C@ @](O)(C#C)
22 d-Norgestrel 1.0 [C@ @](C)(CC[C@ @H]3([C@ @](C)(CC1)2))1)1)3
O=C1CC[C@ @H]([C@@H](CC[C@@H]([C@H](C
23 Etliisterone 1.0 C[C@ @](O)(C#C)[C@](CC(=C)[C@ @H]23)(CC)4)4 )2)C1)3
17-Ethinyl-l 1-methylen O[C@H](CCtC@ @H]([C@ @H](CC[C@ @H]([C@H]
24 e- 18-methyl- 19-nor-DH 1.0 (CC[C@ @](O)(C#C)[C@](CC(=C)[C@ @H]12)(CC)3 T )3)1)C1)2)1
Compounds with medium binding affinity to SHBG
FC1(F)CC[C@@](C)([C@ @H](CC[C@ @H]([C@H](
3α -Hydroxy-5α
25 1.0 CC[C@ @](O)(C#C)[C@ @](C)(CC[C@@H]23)4)4)2) -h-org-2969 Cl)3
17-Ethinyl-3,3-difluoro- F[C@](C(=O)CtC@@](C)([C@@H](CC[C@@](O)(C
26 1.0 5α -androstan-17β-ol )1)[C@H](CCC2=CC(=O)CC[C@ @](C)22)3)1)32
9α
F[C@](CC[C@@](C)([C@ @H](CC[C@H](O)1)[C@
27 -Fluoro-11-oxo- 17-meth 1.0 H](CCC2=CC(=O)CC[C@ @](C)22)3)1)32 yl-testosterone
O=C1C=C2[C@@H](C)C[C@@H]([C@H](CC[C@H
28 9α -Fluoro-testosterone 1.0 ](O)[C@ @](C)(CC[C@ @H]3([C@ @](C)(CC1)2))1)1 )3 ANN
# Compound SMILES output
O=C1C=C2[C@ @H](C[C@H]3([C@H]([C@H](CC[
29 6α -Methyl-testosterone 1.0 C@@](C)([C@ @H](CC[C@@](OC(=O)CC4)44)5)4) [C@ @](C)(CC1)2)5))3
O=C1C=C2C[C@ @H](C)[C@ @H]([C@H](CC[C@
30 Prorenone 1.0 @](O)(C)[C@ @](C)(CC[C@ @H]3([C@@](C)(CC1)2 »1)1)3
7a, O=C1C=C2C[C@@H](C)[C@@H]([C@H](CC[C@H
31 17-Dimethyl-testosteron 1.0 ](O)[C@ @](C)(CC[C@ @H]3([C@ @](C)(CC1)2))1)1 e )3
O=C1C=C2CC[C@H](C(C=C[C@ @](CC)([C@ @H](
32 7 a-Methyl-testosterone 1.0 CC[C@ @](O)(C#C)3)4)3)=C2CC1)4
O=C1C=C2CC[C@@H]([C@H](C[C@@H](O)[C@H
33 R-2323 1.0 ](O)[C@ @](C)(CC[C@ @H]3([C@ @](C)(CC1)2))1)1 )3
16a-Hydroxytestosteron O=C1C=C2CC[C@@H]([C@H](CC[C@ @](O)(C#C)
34 1.0 e [C@ @](C)(CC[C@H]([C@@H](CC1)2)1)2)2)1
O=C1C=C2CC[C@ @H]([C@H](CC[C@@](O)(C#C)
35 Norethindrone 1.0 [C@](CC(=C)[C@H]([C@@H](CC1)2)1)(CC)2)2)1
17-Ethinyl-l 1-methylen
O=C1C=C2CC[C@ @H]([C@H](CC[C@ @](O)(C)[C
36 e- 18-methyl- 19-nortesto 1.0 @ @](C)(CC[C@ @H]3([C@ @](C)(CC1)2))1)1)3 sterone
O=C1C=C[C@ @](C)(C(=C1)C[C@ @H](C)[C@ @H](
37 17-Methyl-testosterone 1.0 [C@H](CCtC@H](Q)tC@ @](C)(CC[C@ @H]12)3)3) 1)2
O=ClCtC@H](CC[C@ @H]([C@H](CC[C@H](O)[C
7a-Methyl-l-dehydrotes
38 1.0 @@](C)(CC[C@ @H]2([C@](C)3([C@H](C1)CCCC tosterone CCN)))1)1)2)3 la-Aminohexyl-17β-hy O=C1CC[C@ @H]([C@H](C1)C[C@ @H](C)[C@ @H
39 droxy-5a 1.0 ]([C@H](CC[C@H](O)tC@ @](C)(CC[C@H]12)3)3)2 -androstan-3-one )1
O=C1CC[C@ @H]([C@ @H](CC[C@@H]([C@H](C
40 7α-Methyl-19-nor-DHT 1.0 C[C@H](O)[C@@](C)(CC[C@ @H]23)4)4)2)C1)3 ANN
# Compound SMILES output
O=C1CC[C@ @H](C2=CCc3cc(O)ccc3[C@ @H](CC[
41 19-Nor-DHT 1.0 C@](C)11)2)1
O=ClCC[C @ @H]([C@H](CCc2cc(O)ccc2[C@ @H](
42 Equilin 1.0 CC[C@](C)11)2)2)1
O=C 1 CC[C @ @H](c2ccc3cc(O)ccc3c2CC[C @] (C) 11 )
43 Estrone 1.0 1
O[C@ @](C#C)(CC[C@ @H]([C@H](CCCl=Cc2o[nH
44 Equilenin 1.0 0]cc2C[C@@](C)l([C@ @H](CC[C@@](C)12)3))3)l )2
O[C@H](C=ClCCtC@ @H]([C@H](CC[C@ @](O)(C
45 Danazol 1.0 #C)tC@](CC(=C)[C@H]([C@@H](CC2)l)l)(CC)3)3) 1)2
O[C@ @H](CC[C@ @](CXC(=CC[C@ @H]([C@H](C
46 3β-Hydroxy-org-2969 1.0 C[C@@](O)(C)[C@@](C)(CCtC@@H]12)3)3)l)Cl) 2)1
O[C@@H](CC[C@@](C)([C@H](C1)C[C@ @H](C)[
47 17-Methyl-delt5AD 1.0 C@@H]([C@H](CC[C@ @](O)(C)[C@@](C)(CC[C @@H]23)4)4)2)3)1
7α, 17-Dimethyl-5a-andr Oclccc2c(C=C[C@ @H]([C@H](CC[C@H](O)[C@ @
48 1.0 ostane-3β,17βb-diol ](C)(CC[C@H]22)3)3)2)cl
Oclccc2c(cl)CC=Cl[C@H](CC[C@H](O)[C@ @](C)
49 6-Dehydro-estradiol 1.0 (CC[C@H]21)1)1
Oclccc2c(cl)CC[C@ @H]([C@H](CCC[C@ @](C)(C
50 7-Dehydro-estradiol 1.0 C[C@H]21)2)2)1
Compounds with moderate binding affinity to SHBG
O=C1CC[C@ @H]([C@H](CC[C@H](C[C@H](O)CC
51 17-Deoxoestrone 1.0 [C@ @](C)2([C@ @H](CC[C@](C)11)3))2)3)1
O=C1C=C2C=C[C@@H]([C@H](CC[C@H](O)[C@
52 Androsterone 1.0 @](C)(CC[C@H]([C@ @H](CC1)2)1)2)2)1
6-Dehydro- 19-nortestost Oclccc2c(cl)CC[C@ @H]([C@H](C[C@ @H](O)[C@
53 1.0 erone H](O)[C@ @](C)(CC[C@H]21)2)2)1 ANN
# Compound SMILES output
O[C@@H](CC[C@@H](C(=C[C@@H](C)[C@@H](
54 Estriol 1.0 tC@H](CC[C@@](O)(C)[C@@](C)(CC[C@H]12)3)3 )2)C2)1)2
O[C@ @H](CC[C@ @H](C(=C[C@ @H](C)[C@ @H](
7a, 17-DimethyI-delta-5
55 1.0 [C@H](CC[C@@](O)(C#C)[C@@](C)(CC[C@H]12) E 3)3)2)C2)1)2
7α
O=C1C=C2CC[C@@H]([C@H](CC[C@H](C(=O)C)[
56 -Methyl- 17-ethinyl-delta 1.0 C@@](C)(CC[C@@H]3([C@@](C)(CC1)2))1)1)3
-5E
O=C1C=C2CC[C@ @H]([C@H](CC[C@](O)(C(=O)C
57 Progesterone 1.0 )[C@ @](C)(CC[C@ @H]3([C@ @](C)(CC 1)2)) 1)1)3
O=C(C)[C@@](O)(CC[C@ @H]([C@H](CC=ClCtC
17-Hydroxyprogesteron
58 1.0 @ @H](O)CC[C@@](C)1([C@@H](CC[C@ @](C)12) e 3))3)1)2
O=C1C=C2CC[C@ @H]([C@H](CC[C@H](C(=O)CO
17-Hydroxy-5-pregnen-
59 1.0 )[C@ @](C)(C[C@H](O)[C@ @H]3([C@ O](C)(CCl) 3b-ol-20-one 2))1)1)3
O=C1C=C2CC[C@@H]([C@H](CC[C@](O)(C(=O)C
60 Corticosterone 1.0 O)[CO O](C)(C[COH](O)[CO @H]3([C@ O](C)(CCl )2))1)1)3
O=C1C=C2CC[C@@H]([C@H](CC[C@](O)(C(=O)C
61 Cortisol 1.0 O)[CO ©](CXCC(=O)[CΘ ©H]3([CΘ Θ](C)(CC1)2)) 1)1)3
O=C[CO](C[COH](O)[CO ΘH]([C© @H](CCC1=C
62 Cortisone 1.0 C(=O)CC[C@@](C)11)[C@H](CC[C@H](C(=O)CO) 2)3)1)32
63 Aldosterone 1.0 O=ClC[C@H](Oc2cc(O)cc(O)c21)clccc(O)ccl
64 Naringenin 1.0 O=C 1 C(=COc2cc(O)cc(O)c21 )c 1 ccc(O)cc 1
65 Genistein 1.0 Oclccc(ccl)C(C)(C)CC(C)(C)C
66 4-Tert-octylphenol 1.0 Oclccc(ccl)CCCCCCCCC
Oclccc(cclOC)C[C@@H](COC[C@H](Cclccc(O)c(
67 4-Nonylphenol 1.0 OC)cl)l)l ANN
# Compound SMILES output
3 ,4-Divanillyltetrahy dro Oclcccc(cl)C[C@@H](COC[C@H](Cclcccc(O)cl)l)
68 1.0 fur an 1
O=C(Oclccc2c(cl)CC[C@ @H]([C@H](CC[C@H](O)
69 Enterofuran 1.0 [C@ @](C)(CC[C@H]21)2)2)l)clcccccl
O=C1C=C2CC[C@H](C(C=C[C@ @](C)([C@@H](C
70 Estradiol-3-benzoate 1.0 C[C@ @](O)(C)3)4)3)=C2CC1(C)C)4
O[C@ @](C#C)(CC[C@ @H]([C@H](CCC1=CCCC[C
71 R-2956 1.0 @ @H]1([C@@H](C(=C)C[C@ @](CC)12)3))3)1)2
17-Ethinyl- 11 -methylen
Oclccc2c(cl)CC[C@@H]([C@H](CCtC@ @](O)(C#
72 e- 18-methyl-4-estren- 17 1.0 C)[C@ @](C)(CC[C@H]21)2)2)1 β-ol
O=C1C=C2[C@@H](C)C[C@ @H]([C@H](CC[C@](
73 17-Ethinylestradiol 1.0 O)(C(=O)C)[C@ @](C)(CC[C@@H]3([C@@](C)(CC 1)2))1)1)3
17-Hydroxy-6α-methylp O=ClOC[C@H](Cc2ccc(O)c(OC)c2)[C@ @H](Cc2cc
74 1.0 rogesterone c(O)c(OC)c2)l
O=C1CC[C@ @](C)([C@ @H](CC[C@ @H]([C@H](C
75 (-)-Matairesinol 1.0 C[C@ @](O)(CCCN)[C@ @](C)(CC[C@ @H]23)4)4)2 )C1)3
17a-Aminopropyl- 17β
76 -hy droxy-5 α-androstan- 1.0 O=ClOc2cc(O)ccc2c2oc3cc(O)ccc3c21 3-one
O=C1CC[C@ @H]([C@H](CC[C@H](C[C@H](O)CC
77 Coumestrol 1.0 [C@ @](C)2([C@ @H](CC[C@](C)11)3))2)3)1 slc2C[C@ @H]([C@ @](C)(CC[C@H](O)[C@](C)(C
78 Etiocholanolone 1.0 O)3)[C@@H](CC(=O)O)c2[nH0]clN[S+2]([O-])([O-] )C)3
Compounds with no known binding affinity to SHBG slcc[nH0]clNC(=O)C[C@ @H]([C@H](O)CC[C@@
79 264,368 0.0 H]([C@ @](C)(CC[C@H](O)[C@ @](C)(CO)1)2)1)2 slc[nH0][nH0]clNC(=O)C[C@ @H]([C@H](O)CC[C
80 283,376 0.0 @ @H]([C@@](C)(CC[C@H](O)[C@@](C)(CO)1)2) 1)2 ANN
# Compound SMILES output
81 283,399 0.0 Oc 1 ccc(N=Cc2oc(C)cc2)cc 1
O(C)clccc(ccl)C=Nclcccc2c(N=Cc3ccc(OC)cc3)cccc
82 5,104,460 0.0 12
83 5,105,230 0.0 O=ClN(OCc2ccccc2)C(=O)c2ccccc21
84 5,136,090 0.0 BrclccccclCSCC(=O)NN=Cclccc(OC)ccl
85 5,185,880 0.0 Iclcccc(cl)C(=O)NC(=S)Nclccc(OC)c(Cl)cl
86 6,412,220 0.0 CIc 1 ccc(NC(=O)c2cccc(c2)C(=O)Nc2ccc(Cl)cc2)cc 1
87 5,349,330 0.0 O=C 1 CC(CC(=O)C 1 C(=O)C)c 1 ccccc 1 c(cl)C(=O)Nclccc(N2CCN(CC2)C(=O)C
Figure imgf000050_0001
Brclccc2N(Cc3ccccc3)C(=O)C(O)(CC(=O)c3ccc(OC)
89 7,628,160 0.0 c(OC)c3)c2cl
S=ClN(c2cccc(c2)C(=O)C)C(=O)[C@@H](NlCclccc
90 7,420,500 0.0 (OC)ccl)CC(=O)Nclcccccl
91 7,356,440 0.0 SlCCN(CCl)Cclccc([N+](=O)[O-])ccl
92 5,257,950 0.0 [O-][N+]l=C(N=C(c2ccccc2)Cl(C)C)clccccclOC
93 5,341,830 0.0 O=C(NN=ClCC(NC(C)(C)Cl)(C)C)clccccclO
94 5,467,400 0.0 Clclccc(CCNCc2ccc(OC)cc2)c(Cl)cl
95 5,528,020 0.0 ClC(Cl)(Cl)[C@ @H](NCclocccl)NC(=O)Cclcccccl
96 5,529,650 0.0 Brclccc(NCc2occc2)ccl
O=C(NN=Cclcccc2ccc[nH0]c21)[C@@H](O)clccccc
97 5,530,840 0.0 1
O=ClNC(C)=C(C(=O)NCc2ccccc2)[C@ @H](Nl)clcc
98 5,763,650 0.0 c(OCCC)c(OCC)cl
99 7,581,570 0.0 O=C(NCc 1 c [nH0]ccc l)c 1 cc(OC)cc(OC)c 1
Brclcccc(cl)C(=O)NC(=S)Nclcccc(clC)cloc2ccc[nH
100 5,658,450 0.0 0]c2[nH0]l
101 6,393,870 0.0 Brclccc(OCC(=O)Nc2sc3cc(OC)ccc3[nH0]2)ccl
102 5,847,360 0.0 Clclccc(cclNC(=O)[C@ @H](SCclcccccl)C)C(F)(F)F
103 7,576,330 0.0 Clclcc([nH0]2[nH0]c3ccc(N)cc3[nH0]2)ccclOC
O=C(NC(C)C)C12C[C@ @H](C[C@ @H](CC(C3)(C2
104 6,019,380 0.0 )c2ccc(C)cc2)Cl)3
105 6,044,690 0.0 O=C(NCC(C)C)clc(C)c([nH0]c2ccccc21)clcccccl ANN
# Compound SMILES output slc(cc(clNC(=O)C[C@H](NclccccclNCl=O)l)C(=O
106 6,106,680 0.0 )OCC)CC
107 6,120,600 0.0 Clclcc(OC)c(NC(=S)NCc2cccc(OC)c2)cclOC
108 7,649,430 0.0 Iclccc(Cl)c(cl)C(=O)NC(=S)NclccccclC(F)(F)F
109 6,618,210 0.0 O=C(NclccccclOC)Cl(CCCCl)clcccccl
Clclccc(ccl)cloc([nH0][nH0]l)Cl=Cc2ccc(N(CC)CC
110 6,621,900 0.0 )cc2OCl=O
Clclsc(Cl)c(cl)C(=O)CScl[nH0]c[nH0]c2sc(C)c(C)c2
111 6,625,660 0.0 1
O=C(Oclccc(ccl)C(=O)N[C@ @H](CC(C)C)C(=O)O
112 6,684,510 0.0 C)C
113 6,788,540 0.0 FclccccclNC(=O)C(=O)NCCOC=C
114 6,940,820 0.0 Brc 1 ccc(cc 1 )C(=O)CNc 1 cc(C)ccc 1 C
115 7,076,670 0.0 O=C(Nclcccc(C)cl)CCCC(=O)Nclcccc(C)cl
116 7,272,580 0.0 Brclccc(NC(=O)c2cc(OC)c(OC)cc2[N+](=O)[O-])ccl
117 5,863,030 0.0 S(CC(=O)N)cl [nH0]c2ccccc2c(C)cl
Brclccc(OCC(=O)O)c(cl)[C@H](NC(=S)NC(C)=ClC
118 7,368,620 0.0
(=o)C)i
Iclc([nH0][nH0](clC)C(=O)cloc(ccl)COclccccclOC
119 7,462,560 0.0 )C
Fclcccc(NC(=O)[C@@H](CC=CC[C@H](C(=O)O)2)
120 7,510,330 0.0 2)clC
Clclc2N=C(C=C([nH0]2[nH0]clC(=O)NCclocccl)C(
121 7,760,360 0.0 F)(F)F)clccc(OC)ccl
122 5,817,870 0.0 Brcloc(ccl)C(=O)NC(=S)NlCCCCl
123 7,678,420 0.0 Clclccc(ccl)C(=O)NC(=S)N(CC)CC
124 7,680,140 0.0 slc([nH0][nH0]clCOC)NC(=O)COCC
125 7,680,400 0.0 S(CCNC(=O)clccc(C)c([N+](=O)tO-])cl)clccc(C)ccl
Clclcc([S+2]([O-])([O-])C(F)(F)F)cc([S+2]([O-])([O-]
126 6,384,810 0.0 )C(F)(F)F)cl
127 7,744,740 0.0 O=ClN(OC(=O)c2ccccc2OCC)C(=O)c2ccccc21
128 7,782,500 0.0 S=C(NClCCCCl)N[C@@H](C)clccc(OC)c(OC)cl
129 7,797,200 0.0 FC(F)(F)CICCCCCINC(^O)NCICCCCCCCI ANN
# Compound SMILES output
130 7,802,020 0.0 Brclcc(cc(OC)clOCclccccclF)CNclccc(O)ccl
131 7,852,940 0.0 Oclccc(N=Cc2ccccc2O)ccl
O=[N+]([O-])clcccc(cl)C(=O)Nclcccc(cl)cloc2ccc(C
132 5,105,180 0.0 )cc2[nH0]l
133 5,255,280 0.0 s 1 c(SC)c(COCC)c(COCC)cl SC
134 5,119,180 0.0 Clclccc(C=C2OC(=S)N(C)C2=O)c(Cl)cl
O=ClOC(=Nc2cccccl2)clccc2c(cl)C(=O)N(clccc(C)
135 5,162,510 0.0 c(C)cl)C2=O
136 5,187,230 0.0 FC(F)(F)clcccc(NC(=O)C23CC4CC(CC(C4)C3)C2)cl
[nHO] 1 [nH0]c([nH0][nH0] 1C12CC3CC(CC(C3)C2)C1
137 5,261,270 0.0 )clcccccl
138 5,316,820 0.0 OlCCOCl(C[nH0]lccc2cccccl2)clcccccl
139 5,322,880 0.0 Brclccc(ccl)cloc(ccl)C=ClC(=O)NC(=S)NCl=O
140 5,377,940 0.0 Oclc(C)cc(C)c2ccc([nH0]cl2)C=CclccccclN
141 5,478,950 0.0 O=ClCC(C)(C)CC(=O)Cl=CNCCclccccclOC
O=[N+]([O-])clccc(ccl)C=NNC(=O)NC12CC3CC(C
142 5,483,070 0.0 C(C3)C2)C1
143 5,548,360 0.0 O(CCCN[C@ @H](C)CC)clccccclOC(C)C
Clclccc(SCc2ccc(cc2)C(=O)NC[C@ @H](OCCC2)2)c
144 7,317,740 0.0 cl
145 5,632,260 0.0 S(CC(=O)Nclcccc(OCCCC)cl)clcccc2ccc[nH0]cl2
146 5,653,560 0.0 [nHO] 1 [nH0](Cc2ccccc2)c2ccccc2cl
147 5,661,530 0.0 O=C(O)CNCl=NC(C)(C)Cc2clccclcccccl2
148 5,674,920 0.0 [nHO]lccc[nHO]clNClCCCCCCl
149 5,691,430 0.0 [nH0]lc2[nH0](C=CC=C2)cclclccc(ccl)CC
150 5,691,500 0.0 slc2CCCCc2c(clNC(=O)Cclcccccl)C(=O)OC
O=ClCC(C)(C)CC=2NC(C)=C(C(=O)OC)[C@@H](c
151 5,702,110 0.0 3ccc(OC(=O)C)c(OCC)c3)Cl=2
152 5,804,410 0.0 N=C(Ncl[nHO]c(C)cc([nHO]l)C)Nclcccc(C)cl
153 5,724,750 0.0 O(C)c lccc(C=NNc2[nH0]c3ccccc3 [nH0]c2C)c(OC)c 1
154 5,750,000 0.0 O=C(NN=C(C)clccccclO)clcc(oclC)C
155 5,783,960 0.0 ClclccccclCScl[nH0]c([nH0][nH]l)clccc(OC)ccl
156 5,924,020 0.0 S(Ccloc(ccl)C(=O)O)CC(=O)O ANN
# Compound SMILES output
Br.SlC=C[nH0]2cl[nH0]c(C)c2cl[nH0]c(scl)Ncl[nH
157 5,926,400 0.0 0]ccc[nH0]l
O=C(Nc 1 cccc(NC(=O)C(C)(c2ccccc2)c2ccccc2)c 1 )c 1 c
158 5,931,470 0.0 cccc 1
159 6,028,810 0.0 Brclccc(OCC(=O)ON=C(N)c2cc[nH0]cc2)cclC
160 6,093,220 0.0 O=C(NCclocccl)C(=O)NclccccclOCC
161 6,197,180 0.0 O=C(N[C@H](C(=O)Nclcccc(cl)CC)C(C)C)clcccccl
O=ClCC(C)(C)CC=2Nc3cc(ccc3N[C@H](C=21)clcc
162 7,785,350 0.0 c(O)cc 1 )C(=O)c 1 ccccc 1
Brclccc(ccl)[C@H](NC(=O)CC[C@H]([N+](=O)tO-]
163 6,383,970 0.0 )1)1
164 6,433,490 0.0 S=C(Nclccc(C)ccl)NC(=O)clccc(ccl)clcccccl
165 6,596,190 0.0 Clclcccc(cl)cloc([nH0][nH0]l)Cl=Cc2ccccc2OCl=O
166 6,629,220 0.0 S=C(NCclccc(OC)ccl)Nclcc(OC)cc(OC)cl
Clclccc(OCC(=O)[nH0]2[nH0][nH0]c3ccccc23)c(C)c
167 6,925,030 0.0 1
168 7,251,940 0.0 Clclccc(NC(=O)CC[C@H](CCC)C(=O)O)c(Cl)cl
169 7,387,600 0.0 Fclccc(ccl)cl[nH0](CCCC)c(ccl)CCC(=O)O
Brclccc(OCC(=O)Nc2[nH0][nH0][nH0]([nH0]2)CCC)
170 7,569,870 0.0 cc 1
Slc2[nH0]c3cc(C)c(C)cc3[nH0]2C(=O)Cl=Cclcccc2c
171 7,572,450 0.0 cccc21
172 5,648,940 0.0 slcc([nH0]clC)clccc(NC(=O)c2ccccc2F)ccl
O=C(NC[C@@H](OCCCl)l)clccc(NC(=O)C2(CCO
173 7,645,140 0.0 CC2)c2ccc(OC)cc2)ccl
174 7,661,160 0.0 0=C(N(CC)clcccccl)cloc2cc(C)c(C)cc2clC
175 7,679,420 0.0 Clclccc2sc(SCC(=O)Nc3ccc4OCCOc4c3)[nH0]c2cl
176 7,701,010 0.0 ClclccccclOCCNC(=O)cl [nH0][nH]c[nH0] 1
177 7,731,150 0.0 O=C(ON=C(N)clcccccl)COclccc(ccl)C(C)C
178 7,733,040 0.0 O(C)clccc(Nc2[nH0]c3ccccc3[nH0]2C)ccl
179 7,762,790 0.0 Clclccc(NC(=O)C(=O)NCCN(C)C)ccl
180 7,805,220 0.0 S=C(Ncl[nH0]c(C)cc(C)cl)Nclsc(C)cclC(=O)OC
181 7,825,890 0.0 S=C([S-])NCCclccc(OC)c(OC)cl.[Na+] ANN
# Compound SMILES output
182 5,107,330 0.0 [S+2]1([O-])([O-])CC[C@H](NC(=O)OCCC)C1
O=C(Nclccc2oc([nH0]c2cl)clcc[nH0]ccl)clcccc(C)c
183 5,155,890 0.0 1
184 5,202,700 0.0 O=C(N)clccc(N=Cc2[nH]ccc2)ccl
185 5,226,770 0.0 O=C(clcccccl)clccc(N=Cc2c3ccccc3[nH]c2C)ccl
Clclcc(N=Cc2ccc(cc2)C=Nc2cc(Cl)c(N)c(Cl)c2)cc(Cl
186 5,226,830 0.0 )clN
O=[N+]([O-])clccc(C=C2N(C)CCC2)c([N+](=O)[O-])
187 5,234,330 0.0 cl
FC(F)(F)clcccc(NC(=O)[C@H]([C@H](O[C@H](C=
188 5,236,760 0.0 C2)[C@ @H](C(=O)O)3)2)3)cl
189 5,278,030 0.0 O=C(NN=Cclc[nH]c2ccccc21)clccc(OC)ccl
190 5,313,660 0.0 O[C@H](COCCC(C)C)C[nH0]lc2ccccc2c2cccccl2
191 5,315,540 0.0 Iclcc(I)c(O)c(cl)C=Nclccc(C)ccl
192 5,321,680 0.0 O=C(NN=Cclcccccl)NN=Cclcccccl
193 5,323,810 0.0 O=[N+]([O-])clcccc(cl)C=NNC(=O)CCCCCCC
O=C(NN=Cclccc2OCOc2cl)Cl[C@ @H](CCCC[C@
194 5,328,830 0.0 @H]21)2
195 5,330,950 0.0 Clclccc([N+](=O)[O-])cclC(=O)NCCclcccccl
196 5,331,410 0.0 olcccclC=NNcl[nHO]c(C)cc([nHO]l)clcccccl
197 5,339,300 0.0 O=C1OC2CCCCC2C(NC(C)(C)C)=C1NC(=O)C
198 5,347,120 0.0 O=ClC=C(Nc2[nH0]c([nH0]c(C)c21)C)clcccccl
O=ClNC(=O)C(=Cc2c[nH0]([nH0]c2c2ccccc2)c2cccc
199 5,359,300 0.0 c2)C(=O)Nl
200 5,361,830 0.0 ClC(CI)(Cl) [C @ @ H](NC(=O)CCC)N(C)Cc 1 ccccc 1
201 5,365,500 0.0 FclccccclC=lOC(=O)C(N=l)=Cclccc(OC(=O)C)ccl
202 5,376,390 0.0 O(C)clccc(ccl)cl[nH0][nH0]c(N)[nH]l
Clclccc(N2C(=O)NC(=O)C(=CNc3ccccc3Cl)C2=O)cc
203 5,377,350 0.0 1
204 5,397,010 0.0 Iclc[nH0]c(N=Cc2cc(Cl)cc(Br)c2O)ccl
205 5,403,360 0.0 CIc 1 CCc(CC 1 )CN(CCO)CCO
206 5,462,170 0.0 O=C(OCC)C(=CNclcccc(C)cl)C(=O)OCC ANN
# Compound SMILES output
O=[N+]([O-])clccc(ccl)C(=O)Nclcccc(cl)cl[nH0]c2[
207 5,468,150 0.0 nH0](C=CC=C2)cl
208 5,471,390 0.0 O=ClNc2ccccc2Cl(cloc(C)ccl)cloc(C)ccl
209 5,477,600 0.0 O=ClCCCc2c3ccccc3[nH0](c21)C(=O)C
210 5,477,970 0.0 O=C(O[C@](C)(C#CCN1CCCC1)C(=C)C)C
211 5,525,120 0.0 [nH]lcc(CCNCc2ccc(C)cc2)c2cccccl2
212 5,527,420 0.0 O=C(NN=C(C)clccc2CCCCc2cl)cl[nH0]ccccl
213 5,528,970 0.0 Brclccc(ccl)C(=O)Nclsc2CCCCCc2clC(=O)N
214 5,529,590 0.0 S(C)cl [nH0]c2cc(OC)ccc2[nH] 1
215 5,540,850 0.0 Clclccc(OCC(=O)NN=CC2CCCCC2)ccl
216 5,544,400 0.0 Br.Clc 1 ccc(cc l)c 1 [nH0]c2SC(=N[nH0]2cl )C
217 5,652,680 0.0 S 1 c2ccccc2C(=O)C 1 =C 1 c2ccccc2NC 1 =0
O=[N+]([O-])clccccclcl[nH0][nH0]c2[nH0]lC(C)(C)
218 5,680,770 0.0 Cclccccc21 slc2C[C@ @H](C)CCc2c(clNC(=O)cl[nH0]cc[nH0]c
219 5,681,460 0.0 1)C(=O)OC
Brclcc(cc(OC)clO)[C@H](NC(=O)NC(clcccccl)=Cl
220 5,702,310 0.0 C(=O)OCC)1
221 5,743,440 0.0 Cl.O[C@H](CNlc2ccccc2N(C)Cl=N)clcccc2ccccc21 slc([N+](=O)[O-])cccl[C@ @H](N(clccccclNC=lCC
222 5,787,610 0.0 (C)(C)CC(=O)C=11)C(=O)C)1
223 5,788,940 0.0 Cl.Fclccc(ccl)[C@ @H](N)CC(=O)O
224 5,789,130 0.0 S(cl[nH0][nH0]c(C)c2cccccl2)cloc2ccccc2[nH0]l
S=C(NCC=C)N[C@@H](C1CN2CC3(C)CN(C1)CC2)
225 5,795,190 0.0 3
FC(F)(F)C=l[nH0]2[nH0]c(cc2N=C2c3ccccc3CCC2=
226 5,819,930 0.0 l)C(=O)NCclocccl
227 5,827,220 0.0 O=C(NCCc 1 CCc(OC)CC 1 )C(=O)NC(C)(C)C
228 5,919,410 0.0 O(C)cl[nH0]cc([nH0]2c(C)ccc2c2ccccc2)ccl
229 5,931,390 0.0 Cl.N=lCCN2c3ccccc3N(Cc3ccc(C)cc3)C=12
230 6,033,320 0.0 Clclccc2[nH]c3c(CCCC3=O)c2cl
231 6,043,150 0.0 O=C(NclccccclCC=O)Nclcccc(C)clC)Cclcccccl
232 6,083,780 0.0 Clcl ccc(ccl)cl [nH0]c(OCC)c2ccccc2[nH0] 1 ANN
# Compound SMILES output
233 6,098,220 0.0 O=C(O)C[nH0]lc2[nH0]c3ccccc3[nH0]c2c2cccccl2
234 6,113,080 0.0 O=C(Nclccc(OC)ccl)ClCCCCCl
235 6,161,440 0.0 Clclc[nH0]c(NC(=O)CC(C)C)ccl
236 6,169,330 0.0 O=C(Nclccc(ccl)[C@@H](C)CC)CC(C)C
Brc 1 ccccc 1 C(=O)NC(=S)Nc 1 sc2CCCCc2c 1 C(=O)OC
237 6,203,970 0.0 C
O=ClCCCC=2Nc3ccccc3N(C(=O)CCCC)[C@H](C=2
238 6,384,040 0.0 l)clccccclOC
239 6,391,460 0.0 Clclccc(c(Cl)cl)C(=O)NCCSCclccc(C)ccl
240 6,398,130 0.0 O=C(Nclcccc2cccccl2)clc2CCCCc2oclC
241 6,408,980 0.0 Clclccc(ccl)C(=O)NC(C(C)C)C(C)C
Fclcccc(NC(=O)C2=C(NC=3CC(C)(C)CC(=O)C=3[C
242 6,427,960 0.0 @H](c3ccc(N(C)C)cc3)2)C)cl
243 6,501,900 0.0 O=C1OC(C)(C)CC11CC(OC1=O)(C)C(=O)C
244 6,515,060 0.0 O=C(OCC)C(=O)cl[nH0]2C=CC=Cc2cclC
245 6,535,030 0.0 Brclccc(NC(=O)[C@ @H](C)CCC)c(F)cl
246 6,614,940 0.0 O=C(N[C@ @H](C)CCC)CCclcccccl
247 6,622,490 0.0 O=C([nH0]lc[nH0]c2cccccl2)N(C)clcccccl slc(NC(=O)c2c(F)c(F)c(F)c(F)c2F)c(c(C)clC(=O)N)C
248 6,738,840 0.0
(=o)occ
249 7,242,090 0.0 O=C(OC)Nclccc(ccl)CCCC
CIc 1 CCc(CC 1 )C(=O)CSc 1 [πHO] [nH0]c([nH0] 1 C)COc 1 c
250 7,291,420 0.0 ccc2ccc[nH0]cl2
O=ClCCCc2c3cc(C)ccc3[nH0](C[C@ @H](O)CNCc3
251 7,353,960 0.0 ccccc3)c21
O=[N+]([O-])clccc(ccl)COclccc2c(OC=C(C2=O)c2c
252 7,465,940 0.0 cccc2OC)cl slc(N)[nH0][nH0]clC12C[C@H](CC(C[C@ @H](Cl)
253 7,426,000 0.0 CC1)C2)1
254 7,510,520 0.0 Fclccc(NC(=O)C23CCC(C)(C(=O)C2)C3(C)C)ccl
255 7,519,560 0.0 O=ClOc2ccc3ccccc3c2C=ClC(=O)NCclcccccl
Brclccc(NC(=O)c2oc(cc2)C[nH0]2[nH0]cc(Cl)c2)c2[n
256 7,550,160 0.0 H0]ccccl2 ANN
# Compound SMILES output
257 7,560,990 0.0 S(CclccccclC)CC(=O)Nclcc([N+](=O)[O-])ccclOC slc2CCCCc2c(clNC(=O)CScl[nH0][nH0]c([nH]l)clc
258 7,568,390 0.0 ccccl)C(=O)OCC C=l[nH0]2[nH0]c(C)cc2N=C(C=l)clccc(OC
Figure imgf000057_0001
260 7,579,890 0.0 O=C(NCclocccl)C(=O)NClCCl
261 7,580,620 0.0 O=C(ON=C(N)clccc(C)ccl)clcccc2ccccc21
O=C(Oclccc(Nc2[nH0]c[nH0]c3ccccc23)ccl)clcctnH
262 7,590,410 0.0 0]ccl
263 7,592,300 0.0 O=C(O)c 1 cccc(c 1 )COc 1 ccc(ccl )CO
264 7,609,790 0.0 Fclccc(OCC(=O)N2c3ccccc3CCc3ccccc23)ccl
S(CC(=O)Nclccc2ccccc2cl)Cl=NC(=O)C[C@H](Nl)
265 7,627,120 0.0 c 1 ccccc 1
266 7,645,780 0.0 S=C(NlCCCCl)clcccc([N+](=O)[O-])cl
267 7,654,880 0.0 Cl.O=[N+]([O-])clccc(ccl)Cl=NCCNl
268 7,657,020 0.0 O=C(OCC)C[C@H](N)clccc(OC)c(OCC)cl
FC(F)C=l[nH0]2[nH0]cc(c2N=C(C=l)clcccccl)C(=O
269 7,660,550 0.0 )NC[C@ @H](OCCC1)1
270 7,662,890 0.0 S=C(Nclccc(Nc2ccccc2)ccl)NClCCCCl
S(CC(=O)Nclccc2OCCOc2cl)cl[nH0]c(O)cc([nH0]l)
271 7,671,690 0.0 CCC
272 7,679,640 0.0 Fclccc(OCC(=O)Nc2[nH0]ccc[nH0]2)ccl
273 7,686,020 0.0 S(CC(=O)Nclcccc(cl)C(=O)C)Clc2ccccc2c2ccccc21 slc2CCCCc2c(clNC(=O)cloc2cc(C)c(C)cc2clC)C(=
274 7,687,320 0.0 O)N
275 7,692,140 0.0 Clclcccc(Cl)clCNC[C@ OH](O)C
O=C(C)clccc(NC(=O)C23CCC(C)(c4[nH0]c5ccccc5[n
276 7,693,280 0.0 H0]c42)C3(C)C)ccl
Clclccc(NC(=O)CSc2[nH0]c3ccccc3[nH]2)cclC(=O)
277 7,730,240 0.0 OCC
278 7,737,900 0.0 S(CC(=O)Nclccc(NC(=O)c2occc2)ccl)clcccccl
279 7,738,720 0.0 Clclc2[nH0]c([S+2]([O-])([O-])CC)[nH]c2ccclOCC ANN
# Compound SMILES output
S(Cclccc(ccl)C(=O)Nclccc(ccl)C(=O)C)cl[nH0]cccc
280 7,741,160 0.0 1
O=C(O[C@ @H](CC[C@H](O[C@H](CC[C@H](OC(
281 7,746,720 0.0 =O)C)1)2)1)2)C
O=ClN=C(N[C@ @H](Cl)C(=O)Nclccc(OC)ccl)Ncl
282 7,760,780 0.0 ccc(C)cclC
Natural compounds identified by the pharmacophore-based virtual screening
O=C1OCC2(COC3(C2)C(C)CCC2C(C)(CO)C(O)CCC
283 Compound 3305 0.858
23C)Cl
OCC1(C)C(O)CCC2(C)C1CCC(C)C12OC2(COCC2)C
284 Compound 4944 0.823 Cl
OCC1(C)C(O)CCC2(C)C1CCC(C)C12OC(C)(CC1)C
285 Compound 3838 0.820 O
Oc 1 cc2c(cc 10)C 1 (CC2(C)C)CC(C)(C)c2cc(O)c(O)cc2
286 compound 696 0.815 1
OCC(O)C 1 (OC2(C)CCC3C(C)(C)C(O)CCC3(C)C2CC
287 Compound 5355 0.814 I)C
O=C 1 OCC=C ICCC 1 C(CCC2C(CCCC 12C)(CO)CO)C
288 Compound 5273 0.736 O
289 Compound 2593 0.736 OCC12COC(c3ccc(O)cc3)C(C(C)=CClC)C2C
O=ClC=CC2=NC34N(C2=Cl)C(=CC(clccc(O)ccl)C
290 Compound 1898 0.724 4CCCC3)clcccccl
O=C1OCC=C1C=CC1C(=C)CCC2C1(C)CCC(O)C2(
291 Compound 2099 0.653 C)CO
O=C 1 OCC(O)C 1 =CCC 1 C(=C)CCC2C 1 (C)CCC(0)C2
292 Compound 4426 0.649 (C)CO
293 Compound 2228 0.612 OCC12COC(c3ccc(O)cc3)C(C2)C(C)=CCl
O=C1OCC(O)C1=CCC1C(=C)CCC2C1(C)CCC(O)C2
294 Compound 5607 0.584 (C)CO
O=C(O)CC1(OC2(CC1)C(C)CCC1C(C)(CO)C(O)CC
295 Compound 1824 0.536 C12QCO
296 Compound 2623 0.516 OCC 12COC(c3ccc(O)c(OC)c3)C(C(C)=CC 1 C)C2C
297 Compound 2835 0.423 Oclccc(cclO)CClN(C)CCc2cc(O)c(O)cc21 ANN
# Compound SMILES output
298 Compound 5537 0.408 O=ClC=CC=2C(=Cl)CCl(O)COc3cc(O)ccc3C=21
299 Compound 5525 0.278 Oclccc2c(OCC3(O)Cc4cc(O)c(O)cc4C23)cl
O=C1OCC(O)C1=CCC1C(=C)CCC2C1(C)CCC(O)C2
300 Compound 5098 0.266 (C)CO
301 Compound 1235 0.245 O=C 1 C=C2CC3 (O)COc4c(ccc(O)c4O)C3=C2C=C 10
302 Compound 4819 0.240 OCCNCC(O)CN1CCC2CC(O)C(OC)CC2C1C
303 Compound 479 0.190 OC(COCICCC(CCIOC)C(O)CC)CNICCCCCI
O=C1OC2CC3=CCCC(C)C3(C)C(O)C2C1CN1CCC2C
304 Compound 1683 0.174
Figure imgf000059_0001
O=C1OC2CC(O)C(O)CC2C(=C1)CN1CCC2(OCCO2)CC
305 Compound 2898 0.168 1
306 Compound 5025 0.158 OCClOC([nH0]2c[nH0]c3c2[nH0]c[nH0]c3N)CClO
OCC[N+](C)(C)CC[nH0]lc[nH0]c2cl[nH0]c[nH0]c2
307 Compound 104 0.155
N
308 Compound 685 0.129 O=C(Nc 1 ccc(NC2OC(CO)C(O)C2O)cc 1 )C
309 Compound 6767 0.128 O=C1OC2CC(OC(=O)C)CCC2C(C)=C1CC(O)C
O=C(OClCC2C(C=ClC)C(C)(C)CCCC2(O)C)clccc(
310 Compound 3455 0.127 O)c(OC)cl
311 Compound 3594 0.117 Oclccc(NC2OCC(O)C(O)C2O)ccl
312 Compound 534 0.115 S(Cclc[nH0]c(C)c(O)clCO)Cclc[nH0]c(C)c(O)clCO
OCClOC([nH0]2c[nH0]c3c2[nH0]c[nH0]c3N)C2OC(
313 Compound 3321 0.112 OC12)OCC
S(CC(=O)NlCCc2cc(O)c(OC)cc2ClC)cl[nH0][nH0]c
314 Compound 9894 0.110 [nH0]lC
315 Compound 856 0.109 O=Clc2cc(N)ccc2NC(C)=ClCCC(=O)C
316 Compound 8590 0.109 O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)O
317 Compound 3427 0.109 O=ClOc2cc(O)c(O)cc2C(=Cl)CN(C)CCO
OCClOC([nH0]2c[nH0]c3c2[nH0]c[nH0]c3N)C(O)Cl
318 Compound 510 0.109 O
319 Compound 8136 0.107 O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)OCC
320 Compound 938 0.106 O=C(Cclccc2ccc[nH0]c2cl)clccc(O)cclO
321 Compound 8355 0.106 O=C 1 Oc2cc(O)ccc2C(C)=C 1 CCQ=O)O
322 Compound 8094 0.103 O=C 1 Oc2cc(O)ccc2C(C)=C 1 CCC(=O)OCC ANN
# Compound SMILES output
323 Compound 5636 0.103 O=C(C=Cclc[nH0]cccl)clccc(O)ccl
S(CC(=O)NlCCc2cc(O)c(OC)cc2ClC)cl[nH0]cc[nH0
324 Compound 764 0.103 ]1C
325 Compound 8130 0.102 Clclcc2c(OC(=O)C(CCC(=O)OCC)=C2C)cclO
326 Compound 5597 0.101 O=C(Cclccc(O)ccl)clccc(O)cclO
Brcl [nH0]c2c([nH0]c[nH0]c2N)[nH0] 1C1OC(CO)C(
327 Compound 1371 0.101 O)ClO
328 Compound 1773 0.098 slc(ccclC(N)C(=O)O)CCCC(=O)O
O=C1OC2CC(O)C(O)CC2C(=C1)CN1CCC(CC1)C(=O)O
329 Compound 4676 0.098 CC
330 Compound 894 0.097 O=C(O)CCCNC(=O)C(O)C(C)(C)CO
331 Compound 6956 0.097 O=C(O)C 1 ccc(OCc2ccoc2C(=O)O)cc 1
332 Compound 5652 0.097 O=C(O)clccc(OCc2occc2C(=O)O)ccl
333 Compound 4954 0.096 O=C(O)C 1 cccc(NC(=O)c2cccc(N)c2)c 1
334 Compound 1422 0.096 O=ClOC(C)=CC(O)=ClC(=O)C=Cclccc(O)c(OC)cl
335 Compound 1372 0.096 O=C(Cclcc(oclC)C(=O)OC)clccc(O)cclO
336 Compound 605 0.095 O=C(O)CCNC(=O)clc[nH0]cc(O)cl
337 Compound 1983 0.095 O=C(O)CCNC(=O)clc[nH0]cc(O)cl
338 Compound 886 0.095 O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)NCC(=O)O
339 Compound 1259 0.095 O=C(Cc 1 oc(cc 1 )C(=O)OC)c 1 c(O)cc(O)cc 1 C
340 Compound 648 0.095 O=C(O)CCNC(=O)clccc(O)ccl
341 Compound 1924 0.094 O=C(Cc 1 oc(cc 1 )C(=O)OC)c 1 ccc(O)cc 10
342 Compound 9999 0.094 O=C1OC2CC(O)CCC2C(C)=C1CCC(=O)NCC(=O)O
343 Compound 325 0.093 O=C1OC2CC(O)CCC2C(C)=C1CCC(=O)NC(C)C(=O)O
344 Compound 4856 0.093 Clclcc2c(OC(=O)C(CCC(=O)NCC(=O)O)=C2C)cclO
O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)NC(C)C(=O
345 Compound 329 0.093 )O
346 Compound 5948 0.093 O=C(O)CCC(=O)Nclcc2c([nH]cc2CCN)c(OC)cl
O=C1OC2CC(O)CCC2C(C)=C1CC(=O)N1CCC(CC1)C(
347 Compound 1044 0.092 =0)0
348 Compound 1637 0.092 O=C 1 Oc2cc(O)ccc2C(C)=C 1 CC(=O)NCC(=O)O
O=ClC=C2Oc3c(c(O)c(C)c(O)c3C(=O)C)C2(C)C(=O
349 Compound 5605 0.092 )C1C(=O)C ANN
# Compound SMILES output
Clclcc2c(OC(=O)C(CCC(=O)NCCC(=O)O)=C2C)ccl
350 Compound 2886 0.092 O
CIc 1 cc2c(OC(=O)C(CCC(=O)NC(C)C(=O)O)=C2C)cc
351 Compound 1913 0.092 IO
O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)NCCCC(=O
352 Compound 1375 0.091 )O
353 Compound 4266 0.091 O=C1OC2CC(O)CC(O)C2C(C)=C1CC(=O)NCC(=O)O
354 Compound 3767 0.091 O=C1OC2C(O)C(O)CCC2C(C)=C1CC(=O)NCC(=O)O
355 Compound 257 0.091 O=Cl Oc2cc(O)ccc2C(C)=C 1CCC(=O)N(C)CC(=O)O
356 Compound 728 0.091 O=C1OC2CC(O)CCC2C(C)=C1CC(=O)NCCC(=O)O
Clclcc2c(OC(=O)C(CCC(=O)NCCCC(=O)O)=C2C)cc
357 Compound 4587 0.091 IO
358 Compound 1748 0.091 O=C1OC2CC(O)CCC2C(C)=C1CC(=O)N(C)CC(=O)O
O=C 1 Oc2cc(O)ccc2C(C)=C 1 CCC(=O)N 1 CCCC 1 C(=
359 Compound 365 0.091 O)O
O=C1OC2C(C)C(O)CCC2C(C)=C1CC(=O)N(C)CC(=O)
360 Compound 210 0.090 O
O=C1OC2C(C)C(O)CCC2C(C)=C1CC(=O)NC(C)C(=O)
361 Compound 1359 0.090 O
Clclcc2c(OC(=O)C(CC(=O)NCCCC(=O)O)=C2C)ccl
362 Compound 2913 0.090 O
O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)NC(CC)C(=
363 Compound 9797 0.090 O)O
Clclcc2c(OC(=O)C(CC(=O)N3CCC(CC3)C(=O)O)=C
364 Compound 3615 0.090 2C)cclO
365 Compound 823 0.090 O=C1OC2CC(O)CCC2C(C)=C1CC(=O)NC(CC)C(=O)O
O=C1OC2C(C)C(O)CCC2C(C)=C1CC(=O)NCCCC(=O)
366 Compound 1079 0.090 O
O=C1OC2C(C)C(O)CCC2C(C)=C1CCC(=O)N(C)CC(=O
367 Compound 972 0.090 )O
Clclcc2c(OC(=O)C(CCC(=O)NC(CC)C(=O)O)=C2C)
368 Compound 2838 0.090 cclO ANN
# Compound SMILES output
O=C1OC2C(O)C(O)CCC2C(C)=C1CC(=O)NC(C)C(=O)
369 Compound 4798 0.090 O
370 Compound 1018 0.090 O=C1OC2CC(O)CCC2C(C)=C1CCC(=O)NCCC(=O)O
O=[N+]([O-])clcc(ccclO)C=CC(=O)C=lC(=O)OC(C)
371 Compound 298 0.090 =CC=1O
Clclcc2c(OC(=O)C(CC(=O)NC(C)C(=O)O)=C2C)ccl
372 Compound 3503 0.090 O
Clclcc2c(OC(=O)C(CCC(=O)NC(CCC)C(=O)O)=C2
373 Compound 3761 0.089 C)cclO
374 Compound 9808 0.089 O=C1OC2CC(O)CCC2C(C)=C1CC(=O)NC(C)C(=O)O
O=C1OC2CC(O)CCC2C(C)=C1CC(=O)NC(CCCC)C(=O
375 Compound 9765 0.089 )O
O=C1OC2CC(O)CC(O)C2C(C)=C1CC(=O)NC(C)C(=O)
376 Compound 4176 0.089 O
O=C 1 Oc2cc(O)cc(O)c2C(C)=C 1 CC(=O)NCCCC(=O)
377 Compound 2987 0.089 O
O=C1OC2C(C)C(O)CCC2C(C)=C1CC(=O)NC(CC)C(=O
378 Compound 393 0.089 )O
379 Compound 1546 0.089 O=C1OC2C(C)C(O)CCC2C(C)=C1CC(=O)NCCC(=O)O
O=C 1 Oc2cc(O)cc(O)c2C(C)=C 1 CC(=O)NC(CC)C(=O
380 Compound 4138 0.089 )O
381 Compound 4478 0.089 CIc 1 cc2c(OC(=O)C(CC(=O)NCC(=O)O)=C2C)cc 10
382 Compound 2750 0.088 O=C1OC2CC(O)CC(O)C2C(C)=C1CC(=O)NCCC(=O)O
O=C1OC2C(O)C(O)CCC2C(C)=C1CC(=O)NC(CC)C(=O
383 Compound 4675 0.088 )O
Clclcc2c(OC(=O)C(CC(=O)N3CCCC3C(=O)O)=C2C
384 Compound 2822 0.088 )cclθ
385 Compound 4731 0.088 O=C1OC2C(O)C(O)CCC2C(C)=C1CC(=O)NCCC(=O)O
386 Compound 1019 0.088 [S+2]([O-])([O-])([O-])CCNC(=O)clc[nH+]cc(O)cl
O=C1OC2CC(O)CC(O)C2C(C)=C1CC(=O)N1CCCC1C(
387 Compound 3259 0.087 =0)0
388 Compound 763 0.087 O=C(N)clc[nH0+](cccl)CC(=O)clccc(O)cclO For screening and identification of drug-like compounds and potential SHBG ligands, a collection of natural compounds (molecules found in nature or their close synthetic analogues/derivatives) totalling more than 48,000 substances was identified from compound databases. From these, only those with well-defined chemical structures available for purchase in sufficient quantities for subsequent analysis were selected for consideration of typical 'drug-likeness' [21] This subset included 23,836 molecules with molecular weights within the 200-500 Dalton molecular weight range, possess 1 - 5 hydrogen bond donors (for example OH, NH, SH groups) and 1 - 10 hydrogen bonds acceptors (for example O, N, S atoms). They also had less than 8 rotating bonds, 1 - 3 aromatic rings or/and 1 - 5 cyclic systems, a total polar surface area of less than 140 A2, and a hydrophobicity of below logP = 8.0. The corresponding drug-likeness filters were implemented within the MOE (Molecular Operation Environment) package [22]. For each of these 23,836 'drug-like' structures, up to 100 distinct conformations were generated using the Catalyst 4.8 software [23]. The Catalyst 4.8 package was used to generate multiple conformations of known SHBG ligands as well as compounds from the database of natural substances. Default parameters of the Catalyst 4.8 software in combination with FAST algorithm [23], default 20kcal/mol energy range and the maximum number of generated conformation per structure set to 100. The resulting dataset, reflecting the conformational space of the 'drug-like' natural derivatives, was then subjected to pharmacophore-based virtual screening, as described above.
EXAMPLE 2
QSAR Modelling and QSAR hit ranking
The MOE package has been used to optimise all molecular structures [22]. The 'inductive' descriptors have been computed by the MOE custom written scripts for all compounds studied. The ANN training has been created and used by the SNNS package [24]. During the learning phase, a learning rate of 0.8 and a learning update threshold of 0.2 were used, while the input patterns were shuffled. The number of hidden nodes varied from 2 to 20, and 20 independent training and testing runs were conducted for each configuration. The corresponding statistical parameters featured in Tables 10a and 10b represent the values averaged over those 20 training/testing runs.
As for the limits of applicability of the developed QSAR model - since it has been based upon rigorously cross- validated ANN, it is expected to work for any organic molecule, as long as its 34 'inductive' QSAR descriptors (presented in Table 9) fit the range of 'inductive' parameters used for the ANN training.
The identified 105 non-steroidal chemicals were prioritised for experimental testing by using a QSAR approach utilising inductive molecular descriptors [25-27, 28-31]. These descriptors are a novel and distinct group of QSAR descriptors that cover a broad range of bound atoms and molecules whose properties vary in relation to their size; polarizability; electronegativity; compactness; mutual inductive and steric influence, and distribution of electronic density.
Inductive descriptors were used in combination with the method of Artificial Neural Networks (ANN) to rank the 105 non-steroidal compounds selected according to their potential binding-affinity to human SHBG. To build a predictive QSAR model a set of more than 70 compounds known to interact with SHBG (entries 1-78 of Table 8) were assembled, and complied a set of about two hundred chemicals [32] with unknown affinities to SHBG as negative controls for the model (entries 79-282 of Table 8). For all 282 molecules, 28 independent inductive QSAR descriptors, that are characterised in greater detail in Table 9, were calculated. The rational for building a QSAR model for SHBG ligands was such that these 28 molecular parameters could be used as independent variables for describing SHBG binding-affinity. Thus, within the training set, the SHBG binders (entries 1-78) were assigned a dependent parameter with a value 1.0, while the compounds from the negative control set (entries 79-282) were all assigned zero.
To relate the inductive descriptors to the binary (1/0) SHBG binding criteria for the 282 molecules studied, a standard back-propagation ANN configuration consisting of 24 input, 8 hidden and 1 output nodes was employed (Figure 3). For effective training of the ANN, the training set of 188 molecules randomly selected as representing 2/3 of the 282 molecules under investigation were used, In each training run, the remaining 94 compounds under investigation were used as the testing set to assess the predictive ability of the model.
In each of 20 independent runs of the ANN, the corresponding false/true positive- and negative-predictions were estimated using a 0.50 cut-off for the output. In other words, ANN outputs greater than 0.50 were considered as positive predictions. The results demonstrated that the ANN achieved up to 99% accuracy in distinguishing SHBG binders within the training sets and 92% accuracy within the testing sets. The results for various cut-off parameters are shown in Tables 10a) and 10b). These data illustrate that the inductive QSAR descriptors distinguish the compounds in the testing set that bind to SHBG from those that do not.
The ANN-based binary QSAR model created was applied to the 105 molecules identified previously by the pharmacophore model-based search for potential SHBG ligands (entries 283-388 of Table 8). The molecular structures of all 105 molecules were processed to calculate 28 inductive descriptors that were then passed through the pre-trained ANN. The resulting network outputs have been assembled in Table 8 and it can be seen that some candidate molecules are ranked very highly by the model. The corresponding top-ranked substances represent very good candidate ligands of human SHBG, and 22 compounds were selected (molecules from Table 8 with network outputs above 0.1) for purchasing and testing. At the same time, a ligand-protein docking procedure was applied to all 105 pharmacophore-identified compounds to support the lead selection by the QSAR model and to expand the set of selected compounds.
Table 9. Inductive QSAR descriptors used to rank the pharmacophore-identified potential SHBG binders.
QSAR descriptor Definition QSAR descriptor Definition
Sum of all positive
Arithmetic mean of group inductive electronegativities of parameters
Average_EO_Neg Most_Pos_Sigma_mol_i atoms with negative σ*( molecule partial charge →atom) within a molecule Smallest atomic
Arithmetic mean of hardness among electronegativities of
Average_EO_Pos Smallest_Neg_Hardness values for atoms with positive negatively charged partial charge atoms.
Smallest atomic
Arithmetic mean of hardness among
AverageJEϊardness hardnesses of all Smallest Pos_Hardness values for atoms of a molecule positively charged atoms
Smallest value of
Arithmetic mean of atomic steric softnesses of atoms
AverageJVegjSoftness SmaIlest_RsJLmol influence with negative partial
Rs(atom→molecul charge e) in a molecule QSAR descriptor Definition QSAR descriptor Definition
Smallest value of
Arithmetic mean of group steric softnesses of atoms
Average_Pos_Softness Smallest_Rs_moI_i influence with positive partial Rs(molecule→ato charge m) in a molecule
Atomic softness of
Iteratively equalized an atom with the
EO_Equalized electronegativity of a Softness_of_Most_Neg most negative molecule charge
Atomic softness of
Molecular softness - an atom with the
GIobaLSoftness sum of constituent Softness_of_Most_Pos most positive atomic softnesses charge
Largest atomic Sum of hardnesses hardness among of atoms with
Largest_Neg_Hardness Sum_Neg_Hardness values for negatively negative partial charged atoms charge
Largest atomic Sum of hardnesses hardness among of atoms with
Largest_Pos_Hardness Sum_Pos_Hardness values for positively positive partial charged atoms charge
Sum of absolute
Largest value of steric values of partial influence
Largest_Rs_mol_i Total_Charge charges on all Rs(molecule→atom) atoms of a in a molecule molecule
Largest negative
Sum of charges on atomic inductive all atoms of a parameter
Most_Neg_Sigma_i_mol Total_Charge_FormaI molecule (formal σ*(atom→molecule) charge of a for atoms in a molecule) molecule QSAR descriptor Definition QSAR descriptor Definition
Largest (by absolute value) negative group Sum of softnesses inductive parameter of atoms with Most_Neg_Sigraa_mol_i Total JVeg_Softness σ*(molecule→atom) negative partial for atoms in a charge molecule
Largest partial charge Sum of softnesses among values for of atoms with Most_Pos_Charge TotalJPos_Softness positively charged positive partial atoms charge
Largest positive
Sum of inductive atomic inductive parameters parameter Most_Pos_Sigma_i_mol Total_Sigma_mol_i σ*(molecule→ato σ*(atom→molecule) m) for all atoms for atoms in a within a molecule molecule
Table 10a) and Table 10b). Parameters of sensitivity, specificity, accuracy and positive predictive value for the developed ANN-based QSAR models on the training (a) and testing (b) sets with the varying threshold parameters. 10a)
Training on 66 % of the data
Cut-off
Specificity Sensitivity Accuracy PPV
0.5/0.5 1 0.98 0.999 0.999
0.6/0.4 0.993 0.9107 0.970 0.981
0.7/0.3 0.979 0.892 0.955 0.943
10b)
Testing on 33 % of the data
Cut-off
Specificity Sensitivity Accuracy PPV
0.5/0.5 0.95 0.840 0.917 0.875
0.6/0.4 0.95 0.840 0.917 0.875
0.7/0.3 0.95 0.840 0.917 0.875 EXAMPLE 3
Docking and Structure-based virtual screening
The Maestro suite [33] was used to prepare protein structures for docking, which was conducted using the Glide 2.7 program [34] with default settings. The protein charges were assigned by the MMFF94 molecular mechanics force field method [35] and the binding pocket of SHBG was identified as 6.5A surrounding the natural ligand DHT from the crystal structure 1D2S.
Several human SHBG crystal structures have been solved and deployed to the Protein Data Bank [36]. These include co-crystal structures of the protein with different steroid ligands, as well as steroid-binding data for numerous human SHBG mutants that can be exploited in the structure-based design of high affinity SHBG ligands [5,6,37,38]. These published crystal structures also demonstrate that SHBG binds androgen and estrogen molecules in different (opposite) orientations (Figure 4), and show that there are two major hydrogen bonding 'anchors' within the SHBG steroid-binding site: one involving Ser42 residue which binds to the C3-carbonyl group in C19 steroids (androgens) and to the 17β-OH group of Ci8 steroids (estrogens), and another formed by the side chains of Asp85 and Asn82, which binds functional groups at the C3 and Cn positions of C\% and C19 steroids, respectively. These two 'anchors' therefore interact selectively with functional groups of androgen and estrogen molecules with respect to their unique orientations within the SHBG steroid-binding site [38].
A consensus docking strategy was developed utilising four SHBG protein structures - PDB entries 1D2S (co-crystallised with DHT), 1F5F, IKDM and ILHU (co-crystallised with estradiol). All protein structures were pre-processed for docking by removing water molecules and reconstructing hydrogen atoms.
The Glide 2.7 program [34] was used to fit 32 SHBG binders with experimentally determined association constants (correspond to entries 1-8, 10-12, 14-16, 18, 19-21, 28, 32, 33, 38, 41, 44, 46, 51, 53 from Table 8) into the four different crystal structures of SHBG. Based on the docking results for all four structures, an averaged consensus score was derived, which was plotted in Figure 5 against known ligand-SHBG association constants (Ka) taken from the literature [18]. Figure 5 shows the r value for the correlation is above 0.81, indicating that the docking protocol reproduced the experimental protein binding affinities with accuracy. Figure 6 illustrates the docked DHT structure, and shows that it is closely aligned with the known orientation of DHT within the crystal structure (1D2S) of the SHBG steroid-binding site. It was also established that, on average, a compound demonstrates good binding affinity to SHBG when the corresponding docking score is below the -7.5 threshold. Figure 7 illustrates the distribution of the consensus docking scores among known SHBG binders and compounds with no known SHBG binding affinity (molecules 82-151 from Table 8 which have been docked as negative controls).
These results demonstrate that the consensus docking score developed is a useful guide for selecting candidate SHBG ligands for experimental testing. This procedure was therefore applied to 105 pharmacophore-selected non-steroidal structures and docked them into the four crystal structures of SHBG. The established consensus docking scores assembled in Table 11 indicate that this set of 105 compounds might contain a large number of SHBG-binding molecules, with more than half of the candidates scoring below the -7.5 threshold, and several molecules yielding a docking score below -9.0 (Figure 7).
The docking scores also demonstrated that most of the molecules selected using the QSAR ranking could be docked into SHBG steroid-binding site very precisely, and these were selected as prime candidates for testing in a wet assay of their SHBG binding properties. Some compounds
(2-[2-(7-hydroxy-4-methyl-2-oxo-chromen-3-yl)acetyl]aminopropanoic acid (9808), 2-[2-(7-hydroxy-4-methyl-2-oxo-chromen-3-yl)acetyl]aminobutanoic acid (823), methyl 4-[2-(2,4-dihydroxyphenyl)-2-oxo-ethyl]-5-methyl-furan-2-carboxylate (1372), 2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-dibenzofuran-l,3-dione (5605), methyl 5-[2-(2,4-dihydroxyphenyl)-2-oxo-ethyl]furan-2-carboxylate (1924), 4-hydroxy-3-[3-(4-hydroxy-3-nitro-ρhenyl)prop-2-enoyl]-6-methyl-pyran-2-one (298), 4-hydroxy-3-[3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoyl]-6-methyl-pyran-2-one (1422)), which were not identified by the QSAR model, demonstrated very good docking potential (consensus scores below -7.5), and these were also selected for testing. In total, 29 non-steroidal compounds were purchased and subjected to testing (Table 11).
EXAMPLE 4
SHBG Ligand-binding Assay and Testing of SHBG ligand candidates
An established competitive ligand binding assay was used to determine the binding affinities of test compounds to human SHBG in relation to those of testosterone and estradiol [43]. In brief, the assay involved mixing 100 μl aliquots of diluted (1:200) human pregnancy serum containing approximately 1 nM SHBG, which was pre-treated with dextran-coated charcoal (DCC) to remove endogenous steroid ligand, with 100 μl tritium labelled DHT ([3H] DHT) at 10 nM as labelled ligand. For the screening assay, triplicate aliquots (100 μl) of a fixed amount (200 μM) of test compound was added this SHBG/[3H] DHT mixture and incubated overnight at room temperature, followed by a 10 min incubation with 500 μl of a DCC slurry at 00C and centrifugation to separate SHBG-bound from free [3H] DHT. Compounds that displaced more than 35% of the [3H] DHT from the SHBG in this assay were then diluted serially, and triplicate aliquots (100 μl) of known concentrations of test compounds were used to generate complete competition curves by incubation with the SHBG/[3H]DHT mixture, and separation of SHBG-bound from free [3H]DHT, as in the screening assay. The amounts of [3H] DHT bound to SHBG at each concentration of competitor ligand were determined by scintillation spectrophotometry and plotted in relation to the amount of [3H] DHT bound to SHBG at zero concentration of competitor. From the resulting competition curves, IC50 concentrations could be calculated if displacement of more than 50% of [3H] DHT from SHBG was achieved.
The association constants (Ka) have been calculated from the relative binding affinity parameters (RBA) using the following equation: Ka(DHT)/[(l+R)/RBA - R], where K3(DHT) = 0.98 x 109 M"1 is the association constant of the DHT and R (0.05) is the ratio of bound-to-free tritium labelled DHT at 50% displacement in the assay.
The 29 compounds selected by the QSAR ranking and virtual docking experiments were screened for their ability to interact with the SHBG steroid-binding site. The screening assay involved a modification of an established competitive steroid ligand-binding assay that employs tritium-labelled DHT ([3H] DHT) as the radio-labelled ligand as described above. The initial screen of compounds was conducted at a single high concentration (approximately 200 μM). Eight non-steroidal compounds 696, 2593, 2228, 2623, 6767, 8590, 5636 and 5597) displaced 35-95 % of the [3H] DHT from the SHBG steroid-binding site (Table 11). These compounds, and a compound (5098) with no activity in the screening assay (negative control), were then selected for a more detailed analysis of their ability to compete [ H] DHT from the human SHBG steroid-binding site relative to known concentrations of the physiologically most important androgen (testosterone) and estrogen (17β-estradiol). The resulting competitive displacement curves generated using these test compounds (Figure 8) illustrate that their potencies as SHBG ligands are very much in line with their rank potencies obtained in the preliminary screening assay (Table 11), with the most effective competitors being 5597, 2623, and 2593. For these compounds an IC50 could be calculated from the plot shown in Figure 8. These IC50 values (5597 = 13.6 μM; 2623 = 22.4 μM; 2593 = 124.5 μM) could then be compared with those of testosterone (15.6 nM) and estradiol (61.6 nM). These data provide a measure of the relative binding affinities (RBAs) of the top three test compounds as compared to testosterone or estradiol, and their RBAs are shown in Table 12 along with their structural alignments against those of testosterone or estradiol produced by the MOE Flexible alignment routine [22].
Although small differences in the association rate constants (K3) for testosterone and estradiol have been reported, they have been determined previously [18] under the same conditions (i.e. at 4°C) as those employed in this study, and are as follows: testosterone (Ka = 1.1 x 109 M"1); estradiol (Ka= 6 x 108 M"1). K3 values for the most active test compounds are estimated based on their RBA values (Table 12).
Table 11. Non-steroidal substances selected for experimental testing based on their QSAR ranking (column 2) and/or consensus docking score (column 3). Columns 4 and 5 reflect the calculated ligand-SHBG association constants and experimentally determined percentage of [3H] DHT substitution in the SHBG binding assay, respectively. A "*"indicates those compounds which satisfy QSAR activity test (column 3), compounds potentially active according to the docking (column 4) and most active chemicals resulting in more than 35% reduction of the radio labelled DHT (column 5).
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Table 12. Structures of the three most active non-steroidal lead compounds superimposed by the flexible alignment with DHT molecule (second column) and an estrogen molecule (third column) and the corresponding values of the SHBG dissociation constants and RBA parameters. Structures of the steroids are labelled in red, the structures of the identified leads are green.
Figure imgf000076_0001
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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings of this invention that changes and modification may be made thereto without departing from the spirit or scope of the appended claims. All patents, patent applications and publications referred to herein are hereby incorporated by reference.

Claims

What is claimed is:
1. A method for identifying a ligand for sex hormone binding globulin (SHBG), the method comprising combining a protein containing a SHBG binding site with a compound and detecting binding of the compound to the protein, said binding being indicative of the compound being said ligand, and wherein the compound comprises pharmacophore A, B or C or a combination thereof, wherein pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.2 angstroms to about 8.5 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 0.3 angstroms to about 5.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about
1.0 angstroms to about 5.3 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 1.0 angstroms to about 6.2 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 0.9 angstroms to about 5.4 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 1.0 angstroms to about 6.1 angstroms from (c-i), and distanced by about 1.0 angstroms to about 5.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site center being distanced by about 5.4 angstroms to about 10.5 angstroms from (a), and distanced by about 1.2 angstroms to about 5.7 angstroms from (b); d) a hydrogen bond acceptor site having a center, and: d-i) a first acceptor projection point distanced by about 0.8 angstroms to about 5.4 angstroms from the hydrogen bond acceptor site center; and d-ii) a second acceptor projection point distanced by about
1.0 angstroms to about 5.3 angstroms from the hydrogen bond acceptor site center, and distanced by about 0.8 angstroms to about 5.9 angstroms from (d-i), said hydrogen bond acceptor site center being distanced by about 0.8 angstroms to about 7.0 angstroms from (a), distanced by about 6.0 angstroms to about 12.1 angstroms from (b), and distanced by about 8.1 angstroms to about 14.0 angstroms from the hydrogen bond donor/acceptor site center; pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 0.5 angstroms to about 5.2 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 2.5 angstroms to about 9.0 angstroms from (e), and distanced by about 2.0 angstroms to about 7.6 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 5.7 angstroms to about 12.3 angstroms from (e), distanced by about 4.6 angstroms to about 10.9 angstroms from (f), and distanced by about 1.2 angstroms to about 6.7 angstroms from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 0.3 angstroms to about 5.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about 0.4 angstroms to about 5.1 angstroms from the hydrogen bond donor site center, and distanced by about 0.6 angstroms to about 5.9 angstroms from i-i), said hydrogen bond donor site center being distanced by about 1.4 angstroms to about 6.4 angstroms from (e), distanced by about 1.4 angstroms to about 6.6 angstroms from (f), distanced by about 4.9 angstroms to about 10.9 angstroms from (g), and distanced by about 7.9 angstroms to about 14.1 angstroms from the hydrogen bond acceptor site center; pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 1.8 angstroms to about 6.6 angstroms from (j);
1) a third hydrophobic/aromatic site distanced by about 3.9 angstroms to about 8.6 angstroms from Q), and distanced by about 0.7 angstroms to about 5.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 0.4 angstroms to about 5.1 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about 1.1 angstroms to about 5.3 angstroms from the hydrogen bond donor site center, and distanced by about 0.8 angstroms to about 6.0 angstroms from (m-i), said hydrogen bond donor site center being distanced by about 1.1 angstroms to about 6.2 angstroms from Q), distanced by about 3.6 angstroms to about 9.4 angstroms from (k), and distanced by about 6.2 angstroms to about 11.4 angstroms from (1); n) a hydrogen bond acceptor site having a center, and: n-i) a first acceptor projection point distanced by about 0.7 angstroms to about 5.1 angstroms from the hydrogen bond acceptor site center; and n-ii) a second acceptor projection point distanced by about
1.0 angstroms to about 5.4 angstroms from the hydrogen bond acceptor site center, and distanced by about 1.0 angstroms to about 5.5 angstroms from (n-i), said hydrogen bond acceptor site center being distanced by about 6.2 angstroms to about 11.0 angstroms from Q), distanced by about 3.0 angstroms to about 7.7 angstroms from (k), distanced by about 1.0 angstroms to about 5.7 angstroms from (1), and distanced by about 8.6 angstroms to about 13.8 angstroms from the hydrogen bond donor site center.
2. The method of claim 1, wherein the compound comprises pharmacophore A.
3. The method of claim 1, wherein the compound comprises pharmacophore B.
4. The method of claim 1, wherein the compound comprises pharmacophore C.
5. The method of claim 2, wherein pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.4 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about
2.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 2.8 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 2.7 angstroms from (c-ii), and distanced by about 2.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site center being distanced by about 7.6 angstroms from (a), and distanced by about 2.4 angstroms from (b); d) a hydrogen bond acceptor site having a center, and: d-i) a first acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond acceptor site center; and d-ii) a second acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond acceptor site center, and distanced by about 2.6 angstroms from (d-i), said hydrogen bond acceptor site center being distanced by about 3.6 angstroms from (a), distanced by about 8.9 angstroms from (b), and distanced by about 10.9 angstroms from the hydrogen bond donor/acceptor site center.
6. The method of claim 3, wherein pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 2.0 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 5.7 angstroms from (e), and distanced by about 4.4 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 8.9 angstroms from (e), distanced by about 7.6 angstroms from (f), and distanced by about 3.3 angstroms from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (i-i), said hydrogen bond donor site center being distanced by about 3.1 angstroms from (e), distanced by about 3.3 angstroms from (f), distanced by about 7.7 angstroms from (g), and distanced by about 10.9 angstroms from the hydrogen bond acceptor site.
7. The method of claim 4, wherein pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 3.5 angstroms from G);
1) a third hydrophobic/aromatic site distanced by about 5.8 angstroms from (j), and distanced by about 2.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (m-i), said hydrogen bond donor site distanced by about 2.8 angstroms from (j), distanced by about 6.3 angstroms from (k), and distanced by about 8.5 angstroms from (1); n) a hydrogen bond acceptor site having a center: n-i) a first acceptor projection point distanced by about 1.9 angstroms from the hydrogen bond acceptor site center; and n-ii) a second acceptor projection point distanced by about 2.0 angstroms from the hydrogen bond acceptor site center, and distanced by about 2.2 angstroms from (n-i), said hydrogen bond acceptor site distanced by about 8.3 angstroms from (j), distanced by about 4.9 angstroms from (k), distanced by about 2.6 angstroms from (1), and distanced by about 11.0 angstroms from the hydrogen bond donor site center.
8. The method of claim 1, 2, 3, 5 or 6, wherein the compound is a steroid.
9. The method of claim 1, 2 or 5, wherein the compound is a steroid.
10. The method of any one of claims 1 to 7 wherein the compound is not a steroid.
11. The method of any one of claims 1 to 9 wherein the compound is not a compound listed in Table 13.
12. The method of any one of claims 1 to 11 wherein the compound is not a compound listed in Table 14.
13. The method of any one of claims 1 to 12 wherein the protein is SHBG.
14. The method of claim 13, wherein the SHBG is human SHBG.
15. The method of any one of claims 1 to 14 comprising comparing the binding of said compound to said protein to binding of a known SHBG ligand to said protein.
16. The method of claim 15 comprising measuring the affinity of the compound for said protein.
17. A method of altering the binding of a sex hormone to SHBG comprising combining SHBG in the presence of the sex hormone with an effective amount of an SHBG ligand comprising a pharmacophore as defined in any one of claims 1 to 7, providing that the ligand is not listed in Table 13 or 14.
18. The method of claim 17, wherein the ligand competes with the sex hormone for binding to SHBG.
19. The method of claim 17 or 18, wherein the ligand is not a steroid.
20. The method of claim 17, 18 or 19, wherein said combining is performed in vivo in a mammal.
21. The method of claim 20, wherein the mammal is suffering from a condition characterised by a decrease in availability of the sex hormone or which is amenable to treatment by increasing the availability of the sex hormone.
22. The method of claim 17, 18 or 19, wherein said combining is in a biological sample from a mammal.
23. The method of claim 20, 21 or 22, wherein the mammal is a human.
24. Use of a SHBG ligand comprising a pharmacophore as described in any one of claims 1 to 7 for altering binding of a sex hormone to SHBG, providing that the ligand is not listed in Table 13 or 14.
25. Use of a SHBG ligand comprising a pharmacophore as described in any one of claims 1 to 7 for preparation of a medicament for altering binding of a sex hormone to SHBG, providing that the ligand is not listed in Table 13 or 14.
26. The use of claim 24 or 25, wherein the ligand competes with the sex hormone for biding to SHBG.
27. The use of claim 24, 25 or 26, wherein the ligand is not a steroid.
28. The use of any one of claims 24 to 27, for treatment of a condition in a mammal characterised by a decrease in availability of the sex hormone or which is amenable to treatment by increasing the availability of the sex hormone.
29. The use of claim 28, wherein the mammal is a human.
30. A composition comprising a pharmaceutically acceptable carrier and an effective amount of at least one SHBG ligand comprising a pharmacophore as defined in any one of claims 1 to 7, for use in altering binding of a sex hormone to SHBG in a mammal, wherein the ligand is not listed in Table 13 or 14.
31. The composition of claim 30, wherein the ligand is not a steroid.
32. A method, use or composition of any one of claims 17 to 31, or a ligand identified by the method of any one of claims 1 to 16, wherein the ligand has the structure of Formula I:
Figure imgf000087_0001
Formula I wherein A1 is selected from the group consisting of: -OH, -SH, -NH2, and -NHR, where R is a 2 to 7 atom substituent; A7 is selected from the group consisting of: =0, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, and ether-linked aliphatic rings having between 3 and 7 members; A2, A3, A4, A5, A& As, A9, A10, An or A12 are independently selected from the group consisting of an alkyl and a substituted alkyl; and, A3 and A4, or A4 and A5, or A6 and A7, or A7 and As, or A9 and A10, or A10 and A11 may be optionally joined to form a cycloalkyl.
33. A method, use or composition of any one of claims 17 to 31 , or a ligand identified by the method of any one of claims 1 to 16, wherein the ligand has the structure of Formula II:
Figure imgf000087_0002
Formula II wherein Ei, E2, E3, E4, E5, EQ, E9, E10 or En are independently selected from the group consisting of: H, an alkyl, a substituted alkyl, =0, =S, R-C=O, =N-R, NO2, CN, halogen, and SO2R where R is a 2 to 7 atom substituent; and at least one of Ei and E2 is selected from the group consisting of: =0, =S, R-C=O, =N-R, NO2, CN, halogens, and SO2R; E7 and Es are independently selected from the group consisting of: an alkyl, a substituted alkyl, -OH, -SH, -NH2, and -NHR, wherein at least one of E7 and E8 is selected from the group consisting of: -OH, -SH, -NH2, -NHR; and, E4 and E5, or E5 and E6 or E9 and E10, or E10 and En may be optionally joined to form a cycloalkyl group.
34. A method, use or composition of any one of claims 17 to 31 , or a ligand identified by the method of any one of claims 1 to 16, wherein the ligand has the structure of Formula III:
Figure imgf000088_0001
Formula III wherein Bi, B2, B3, B4, B5, Be and B7 are independently selected from the group consisting of: H, an alkyl, a substituted alkyl, -OH, -SH, -NH2, and -NHR, =0, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, and ether-linked aliphatic rings having between 3 and 7 members and R is a 2 to 7 atom substituent, wherein at least one of B5, Bg or B7 is selected from the group consisting of: -OH, -SH, -NH2, and -NHR and at least one of Bi, B2 or B3 is selected from the group consisting of: =0, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, and ether-linked aliphatic rings having between 3 and 7 members; and B5 and Bβ or B6 and B7 may be optionally joined to form a cycloalkyl.
35. A method, use or composition of any one of claims 17 to 31, or a ligand identified by the method of any one of claims 1 to 16, wherein the ligand has the structure of Formula IV:
Figure imgf000089_0001
Formula IV wherein D1 and D2, D3 and D4 are independently selected from the group consisting of =O, =S, R-C=O, =N-R, NO2, CN, halogen, SO2R, ether-linked aliphatic rings having between 3 and 7 members, -OH, -SH, -NH2, and -NHR, where R is a 2 to 7 atom substituent; D5, D6, D7, Dg, D9 and D1O are independently selected from the group consisting of H, an alkyl and a substituted alkyl; and D5 and Oe, or Dg and D7, or D7 and Ds, or Ds and D9, or D9 and D10 may be optionally joined to form a cycloalkyl structure.
36. A method, use or composition of any one of claims 17 to 31, or a ligand identified by the method of any one of claims 1 to 16, wherein the ligand has the structure of Formula V:
Figure imgf000089_0002
Formula V wherein Fi, F2, F3. F4, F5, Fβ, F7, Fs and F9 are independently selected from the group consisting of H, an alkyl, a substituted alkyl, -OH, -SH, -NH2, -NHR, =O, =S, R-C=O, =N-R, NO2, CN, halogen and SO2R, wherein at least one of Fi and F2 is selected from the group consisting of: -OH, -SH, -NH2, and -NHR, and at least one of F3 and F4 is selected from the group consisting of =0, =S, R-C=O, =N-R, NO2, CN, halogen and SO2R, where R is a 2 to 7 atom substituent.
37. A computer implemented method for identifying a candidate ligand for sex hormone binding globulin (SHBG) comprising:
I) providing a data structure comprising data for a plurality of chemical structures identifying a functionality for individual sites within each chemical structure according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic;
II) providing a data structure comprising data for a pharmacophore identifying a functionality for individual sites within the pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and spatial co-ordinates for each site having said functionality; and
III) comparing the data structures of (I) and (II) and identifying a chemical structure in (I) matching the pharmacophore of (II); wherein the pharmacophore is: pharmacophore A containing: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.2 angstroms to about 8.5 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 0.3 angstroms to about 5.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 1.0 angstroms to about 5.3 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 1.0 angstroms to about 6.2 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 0.9 angstroms to about 5.4 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 1.0 angstroms to about 6.1 angstroms from (c-i), and distanced by about 1.0 angstroms to about 5.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site center being distanced by about 5.4 angstroms to about 10.5 angstroms from (a), and distanced by about 1.2 angstroms to about 5.7 angstroms from (b); d) a hydrogen bond acceptor site having a center, and: d-i) a first acceptor projection point distanced by about 0.8 angstroms to about 5.4 angstroms from the hydrogen bond acceptor site center; and d-ii) a second acceptor projection point distanced by about
1.0 angstroms to about 5.3 angstroms from the hydrogen bond acceptor site center, and distanced by about 0.8 angstroms to about 5.9 angstroms from (d-i), said hydrogen bond acceptor site center being distanced by about 0.8 angstroms to about 7.0 angstroms from (a), distanced by about 6.0 angstroms to about 12.1 angstroms from (b), and distanced by about 8.1 angstroms to about 14.0 angstroms from the hydrogen bond donor/acceptor site center; pharmacophore B containing: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 0.5 angstroms to about 5.2 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about
2.5 angstroms to about 9.0 angstroms from (e), and distanced by about 2.0 angstroms to about
7.6 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 5.7 angstroms to about 12.3 angstroms from (e), distanced by about 4.6 angstroms to about 10.9 angstroms from (f), and distanced by about 1.2 angstroms to about 6.7 angstroms- from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 0.3 angstroms to about 5.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about
0.4 angstroms to about 5.1 angstroms from the hydrogen bond donor site center, and distanced by about 0.6 angstroms to about 5.9 angstroms from i-i), said hydrogen bond donor site center being distanced by about
1.4 angstroms to about 6.4 angstroms from (e), distanced by about 1.4 angstroms to about 6.6 angstroms from (f), distanced by about 4.9 angstroms to about 10.9 angstroms from (g), and distanced by about 7.9 angstroms to about 14.1 angstroms from the hydrogen bond acceptor site center; or pharmacophore C containing: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about
1.8 angstroms to about 6.6 angstroms from (j);
1) a third hydrophobic/aromatic site distanced by about
3.9 angstroms to about 8.6 angstroms from (j), and distanced by about 0.7 angstroms to about
5.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 0.4 angstroms to about 5.1 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about
1.1 angstroms to about 5.3 angstroms from the hydrogen bond donor site center, and distanced by about 0.8 angstroms to about 6.0 angstroms from (m-i), said hydrogen bond donor site center being distanced by about
1.1 angstroms to about 6.2 angstroms from (j), distanced by about 3.6 angstroms to about 9.4 angstroms from (k), and distanced by about 6.2 angstroms to about 11.4 angstroms from
(D; n) a hydrogen bond acceptor site having a center, and: n-i) a first acceptor projection point distanced by about 0.7 angstroms to about 5.1 angstroms from the hydrogen bond acceptor site center; and n-ii) a second acceptor projection point distanced by about
1.0 angstroms to about 5.4 angstroms from the hydrogen bond acceptor site center, and distanced by about 1.0 angstroms to about 5.5 angstroms from (n-i), said hydrogen bond acceptor site center being distanced by about
6.2 angstroms to about 11.0 angstroms from (j), distanced by about 3.0 angstroms to about
7.7 angstroms from (k), distanced by about 1.0 angstroms to about 5.7 angstroms from (1), and distanced by about 8.6 angstroms to about 13.8 angstroms from the hydrogen bond donor site center; and wherein the chemical structure so identified is a candidate ligand.
38. The method of claim 37, wherein the pharmacophore is pharmacophore A.
39. The method of claim 38, wherein pharmacophore A contains: a) a first hydrophobic/aromatic site; b) a second hydrophobic/aromatic site distanced by about 5.4 angstroms from (a); c) a hydrogen bond donor/acceptor site having a center, and: c-i) a first donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center; c-ii) a second donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, and distanced by about 2.8 angstroms from (c-i); and c-iii) a third donor/acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond donor/acceptor site center, distanced by about 2.7 angstroms from (c-ii), and distanced by about 2.8 angstroms from (c-ii), said hydrogen bond donor/acceptor site center being distanced by about 7.6 angstroms from (a), and distanced by about 2.4 angstroms from (b); d) a hydrogen bond acceptor site having a center, and: d-i) a first acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond acceptor site center; and . d-ii) a second acceptor projection point distanced by about 2.1 angstroms from the hydrogen bond acceptor site center, and distanced by about 2.6 angstroms from (d-i), said hydrogen bond acceptor site center being distanced by about 3.6 angstroms from (a), distanced by about 8.9 angstroms from (b), and distanced by about 10.9 angstroms from the hydrogen bond donor/acceptor site center.
40. The method of claim 37, wherein the pharmacophore is pharmacophore B.
41. The method of claim 40, wherein pharmacophore B contains: e) a first hydrophobic/aromatic site; f) a second hydrophobic/aromatic site distanced by about 2.0 angstroms from (e); g) a third hydrophobic/aromatic site distanced by about 5.7 angstroms from (e), and distanced by about 4.4 angstroms from (f); h) a hydrogen bond acceptor site distanced by about 8.9 angstroms from (e), distanced by about 7.6 angstroms from (T), and distanced by about 3.3 angstroms from (g); and i) a hydrogen bond donor site having a center, and: i-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and i-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (i-i), said hydrogen bond donor site center being distanced by about 3.1 angstroms from (e), distanced by about 3.3 angstroms from (T), distanced by about 7.7 angstroms from (g), and distanced by about 10.9 angstroms from the hydrogen bond acceptor site.
42. The method of claim 37, wherein the pharmacophore is pharmacophore C.
43. The method of claim 42, wherein pharmacophore C contains: j) a first hydrophobic/aromatic site; k) a second hydrophobic/aromatic site distanced by about 3.5 angstroms from (j);
1) a third hydrophobic/aromatic site distanced by about 5.8 angstroms from (j), and distanced by about 2.6 angstroms from (k); m) a hydrogen bond donor site having a center, and: m-i) a first donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center; and m-ii) a second donor projection point distanced by about 2.0 angstroms from the hydrogen bond donor site center, and distanced by about 2.6 angstroms from (m-i), said hydrogen bond donor site distanced by about 2.8 angstroms from (j), distanced by about 6.3 angstroms from (k), and distanced by about 8.5 angstroms from (1); n) a hydrogen bond acceptor site having a center: n-i) a first acceptor projection point distanced by about 1.9 angstroms from the hydrogen bond acceptor site center; and n-ii) a second acceptor projection point distanced by about 2.0 angstroms from the hydrogen bond acceptor site center, and distanced by about 2.2 angstroms from (n-i), said hydrogen bond acceptor site distanced by about 8.3 angstroms from Q), distanced by about 4.9 angstroms from (k), distanced by about 2.6 angstroms from (1), and distanced by about 11.0 angstroms from the hydrogen bond donor site center.
44. The method of any one of claims 37 to 43 further comprising:
IV) providing a data structure comprising data for the candidate ligand comprising said functionality and co-ordinates for sites within the candidate ligand;
V) providing a data structure comprising data for a sex hormone binding site of SHBG including functionality of sites according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic and spatial co-ordinates for each site having said functionality; and
VI) comparing the data structures of (IV) and (V) to provide a model showing presence or absence of binding of the ligand to the binding site.
45. The method of claim 44, wherein the data structures (IV) and (V) further comprise a functionality for sites having spatial co-ordinates defining the position of a positive charge.
46. The method of claim 44 or 45, wherein data structures (IV) and (V) further comprise co-ordinates defining size and shape of said ligand and said binding site respectively, wherein said model shows presence or absence of fit of said ligand with said binding site.
47. The method of any one of claims 37 to 46 further comprising:
VII) obtaining a compound comprising the chemical structure of candidate ligand;
VIII) combining the compound with a protein containing the sex hormone binding site; and
IX) detecting binding of the compound to the binding site.
48. The method of claim 47, further comprising measuring binding affinity of the compound for the binding site.
49. A data structure embodied in a computer readable medium comprising means for storing a functionality for individual sites within a pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and means for storing spatial co-ordinates for each site having said functionality; wherein the pharmacophore is a pharmacophore as defined in any one of claims 37 to 43
50. A data structure embodied in a computer readable medium comprising fields storing a functionality for individual sites within a pharmacophore according to at least: hydrogen bond donor, hydrogen bond acceptor, aromatic ring, and hydrophobic; and at least one field for storing spatial co-ordinates for each site having said functionality, wherein the pharmacophore is a pharmacophore as defined in any one of claims 37 to 43.
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