WO1997015659A1 - Crystalline frap complex - Google Patents
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- WO1997015659A1 WO1997015659A1 PCT/US1996/016953 US9616953W WO9715659A1 WO 1997015659 A1 WO1997015659 A1 WO 1997015659A1 US 9616953 W US9616953 W US 9616953W WO 9715659 A1 WO9715659 A1 WO 9715659A1
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- C—CHEMISTRY; METALLURGY
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
Definitions
- the invention relates to a complex, in crystalline form, of two proteins, FKBP12 and the
- FRB domain of FRAP in association with rapamycin, a small organic molecule to which the proteins bind.
- the crystalline form of this ternary complex is particularly useful for the determination of the three-dimensional structure of the complex at the atomic level.
- the three dimensional structure provides information useful for the design of pharmaceutical compositions which inhibit the biological function of proteins such as FRAP which contain an FRB domain, particularly those biological functions mediated by molecular interactions involving rapamycin or other compounds capable of binding to an FRB domain.
- Rapamycin (sometimes called sirolimus) was first described in 1975 as an antifungal agent isolated from Streptomyces hygroscopicus (Vezina, 1975; Sehgal, 1975).
- FK506 (sometimes called tacrolimus) was characterized as a potent immunosuppressive agent (Tanaka, 1987), and shortly thereafter, rapamycin was also shown to have potent immunosuppressive activity.
- FK506 inhibits the T cell receptor (TCR) signal and prevents activation of a resting helper T cell. Rapamycin inhibits the autocrine signaling pathway involving inter leukin-2 (I -2) and the IL-2 receptor (IL-2R). These latter signals commit the cell to a program of cell division by communicating with the components of the cell cycle machinery necessary for DNA replication.
- I -2 inter leukin-2
- IL-2R IL-2 receptor
- FK506 and rapamycin are potentially useful in the treatment of human disease.
- FK506 has been approved by the FDA for use in treating the rejection of transplanted organs.
- a similar use has been envisioned for rapamycin, and its demonstrated activity in organ transplantation and autoimmune animal models indicate a high clinical potential.
- Rapamycin has been shown to have antitumor activity against B16 melanocarcinoma, colon 26 tumor, EM ependymoblastoma, CD8F1 mammary and colon 38 murine tumors (Sehgal, 1993).
- Rapamycin has also shown immunosuppressive activity in assays to measure prevention of development of autoimmune adjuvant arthritis, experimental allergic encephalomyelitis and autoimmune uveoretinitis in the rat (Sehgal, 1993).
- the biological activity and structural novelty of both rapamycin and FK506 led to a search for their cellular target(s), and the target of both compounds was identified as the plentiful cytoplasmic protein FKBP12 (for FK506 binding protein) of 12 kDa molecular mass. Since FK506 and rapamycin bound to the same target (Kd of 0.4 and 0.2 nM, respectively) and affected different pathways, a new function was attributed to the FKBP12-ligand complex.
- FKBP12-FK506 and FKBP12-rapamycin complexes inhibit the individual components, to bind to and inhibit still other protein targets.
- the FKBP12-FK506 complex inhibits the phosphatase activity of calcineurin, a crucial component of the TCR pathway. Calcineurin is a serine/threonine phosphatase also called PP2B.
- the FKBP12-rapamycin complex inhibits the IL-2R signal by binding to a large (289kDa) protein named FRAP in humans (Brown et al, 1994) or RAFT in rats (Sabatini et al, 1994; Chiu et al, 1994).
- FK506 not involved in binding FKBP12 interacts with calcineurin and inhibits its phosphatase activity.
- FRAP phosphatidylinositol
- ATM for ataxia telngiectasia mutant
- telngiectasia mutant is responsible for a human autosomal hereditary disease characterized by cerebellar degeneration, progressive mental retardation, uneven gait, dilation of blood vessels, immune deficiencies, premature aging and a hundredfold increase in cancer susceptibility (Zakian, 1995). Persons who are heterozygous in ATM are believed to be at elevated risk for cancer. Mutations to TEL1 lead to abnormally short telomeres, and in conjunction with other mutations can lead to sensitivity to X-rays, UV radiation and hydroxyurea.
- DNA-PK is, as the name suggests, a DNA-dependent protein kinase that recognizes damaged DNA, and human cells without DNA-PK activity are radiation sensitive and repair deficient.
- MEC1 is required for both S-M and G2-M checkpoint progression as well as for meiotic recombination in yeast.
- MEC1 is arguably the master checkpoint gene in yeast.
- FRAP is a large protein (2549 amino acid residues), and only a small fraction can be involved in recognizing the FKBP12-rapamycin complex. Fortunately all of these residues are in one domain, and this domain, which is called the FKBP12-rapamycin binding (FRB) domain, is the protein used in this invention. It was identified through tryptic digests of FRAP and independently produced as an 11 kDa soluble protein (Chen et al, 1995)
- This invention centers on the FRB domain of human FRAP and begins with obtaining crystals of human FKBP12-rapamycin-FRB of sufficient quality to determine the three dimensional (tertiary) structure of the complex by X-ray diffraction methods.
- obtaining protein crystals in any case is a somewhat unpredictable art, especially in cases in which the practitioner lacks the guidance of prior successes in preparing and/or crystalizing any closely related proteins.
- Obtaining our first crystals of the ternary complex was therefore itself an unexpected result.
- our data represents the first detailed information available on the three dimensional structure of FRAP or of any of the PIK-related kinases and revealed an unpredicted array of surface features.
- the structure shows — in atomic detail — how a small organic molecule, rapamycin, can be used to hold two proteins, FKBP12 and FRB, in close proximity. As such, this structure contains important lessons for the design of heterodimerizing agents.
- the knowledge obtained concerning the FRB of FRAP can be used to model the tertiary structure of related proteins.
- the structure of renin has been modeled using the tertiary structure of endothiapepsin as a starting point for the derivation.
- Model building of cercarial elastase and tophozoite cysteine protease were each built from known serine and cysteine proteases that have less than 35% sequence identity. The resultant models were used to design inhibitors in the low micromolar range. (Proc. Natl. Acad. Sci. 1993, 90, 3583).
- one object of this invention is to provide a composition, in crystalline form, comprising a protein containing an FRB domain.
- the protein may have a bound ligand or may be part of a complex with a second protein molecule and a shared ligand.
- the crystalline composition may contain a complex containing a first protein having a peptide sequence derived or selected from that of an FKBP12 protein, e.g., human FKBP12; a second protein having a peptide sequence derived or selected from that of an FRB domain of a PIK- related kinase family member, e.g. the FRB domain of human FRAP; and a ligand such as rapamycin which is capable of binding to both proteins to form a ternary complex.
- Such a crystalline composition may contain one or more heavy atoms, e.g., one or more lead, mercury, gold and /or selenium atoms.
- Such a heavy atom derivative may be obtained, for example, by expressing a gene encoding the protein of interest under conditions permitting the incorporation of one or more heavy atom labels (e.g. as in the incorporation of selenomethionine), reacting the protein with a reagent capable of linking a heavy atom to the protein (e.g. trimethyl lead acetate) or soaking a substance containing a heavy atom into the crystals.
- a heavy atom label e.g. as in the incorporation of selenomethionine
- Preferred crystalline compositions of this invention are capable of diffracting x-rays to a resolution of better than about 3.5 A, and more preferably to a resolution of 2.7 A or better, and are useful for determining the three-dimensional structure of the material. (The smaller the number of angstroms, the better the resolution.)
- Crystalline compositions of this invention specifically include those in which the crystals are characterized by the structural coordinates of the FRB protein set forth in the accompanying Appendix I or characterized by coordinates having a root mean square deviation therefrom, with respect to backbone atoms of amino acids listed in Appendix I, of 1.5 A or less. Furthermore, our crystalline compositions include crystals characterized by the structural coordinates of both the FRB and FKBP12 proteins set forth in Appendix I, optionally including a molecule of rapamycin as defined structurally by the accompanying coordinates therefor. Structural coordinates of a crystalline composition of this invention may be stored in a machine-readable form on a machine-readable storage medium, e.g.
- a computer hard drive, diskette, DAT tape, etc. for display as a three-dimensional shape or for other uses involving computer-assisted manipulation of, or computation based on, the structural coordinates or the three-dimensional structures they define.
- data defining the three dimensional structure of a composition of this invention or a portion thereof containing an FRB domain- containing protein of the PIK-related kinase family, or portions or structurally similar homologues of such proteins may be stored in a machine-readable storage medium, and may be displayed as a graphical three-dimensional representation of the protein structure, typically using a computer capable of reading the data from said storage medium and programmed with instructions for creating the representation from such data.
- This invention thus encompasses a machine, such as a computer, having a memory which contains data representing the structural coordinates of a crystalline composition of this invention, e.g. the coordinates set forth in Appendix I, together with additional optional data and instructions for manipulating such data.
- data may be used for a variety of purposes, such as the elucidation of other related structures and drug discovery.
- a first set of such machine readable data may be combined with a second set of machine- readable data using a machine programmed with instructions for using the first data set and the second data set to determine at least a portion of the coordinates corresponding to the second set of machine-readable data.
- the first set of data may comprise a Fourier transform of at least a portion of the coordinates for the complex set forth in Appendix I
- the second data set may comprise X-ray diffraction data of a molecule or molecular complex.
- one of the objects of this invention is to provide three-dimensional structural information on the FRB domain of FRAP, of other members of the PIK-related kinase family which containg homologous FRB domains, and of homologs or variants thereof, preferably in association with a bound ligand or bound ligandrprotein complex (such as FKBP12-rapamycin).
- a bound ligand or bound ligandrprotein complex such as FKBP12-rapamycin.
- molecular replacement uses a molecule having a known structure as a starting point to model the structure of an unknown crystalline sample. This technique is based on the principle that two molecules which have similar structures, orientations and positions in the unit cell diffract similarly. Molecular replacement involves positioning the known structure in the unit cell in the same location and orientation as the unknown structure.
- the atoms of the known structure in the unit cell are used to calculate the structure factors that would result from a hypothetical diffraction experiment. This involves rotating the known structure in the six dimensions (three angular and three spatial dimensions) until alignment of the known structure with the experimental data is achieved. This approximate structure can be fine-tuned to yield a more accurate and often higher resolution structure using various refinement techniques. For instance, the resultant model for the structure defined by the experimental data may be subjected to rigid body refinement in which the model is subjected to limited additional rotation in the six dimensions yielding positioning shifts of under about 5%. The refined model may then be further refined using other known refinement methods.
- Another object of the invention is to provide a method for determining the three- dimensional structure of a protein containing an FRB domain, or a complex of the protein with a ligand therefor, using homology modeling techniques and structural coordinates for a composition of this invention.
- Homology modeling involves constructing a model of an unknown structure using structural coordinates of one or more related proteins, protein domains and/or subdomains. Homology modeling may be conducted by fitting common or homologous portions of the protein or peptide whose three dimensional structure is to be solved to the three dimensional structure of homologous structural elements. Homology modeling can include rebuilding part or all of a three dimensional structure with replacement of amino acids (or other components) by those of the related structure to be solved. The structural coordinates obtained for the related protein or complex may be stored, displayed, manipulated and otherwise used in like fashion as those for the ternary complex of FKBP12-rapamycin-FRB set forth in Appendix I.
- Crystalline compositions of this invention thus provide a starting material, and their three dimensional structure coordinates a point of reference, for use in solving the three-dimensional structure of other proteins containing an FRB domain homologous to that of FRAP, as well as complexes containing such a protein.
- Sequence similarity may be determined using any conventional similarity matrix. (See e.g. Dayhoff,1979; Greer, 1981; and Gonnet, 1992). Proteins containing at least one FRB domain having at least 15% peptide sequence identity or similarity with respect to our FRB, as determined by any of the approaches described above, are considered FRAP homologs for the purpose of this disclosure.
- the three dimensional structure defined by the machine readable data for the FRB domain may be computationally evaluated for its ability to associate with various chemical entities.
- chemical entity refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes.
- a first set of machine-readable data defining the 3-D structure of FRAP or a FRAP homolog, or a portion or complex thereof is combined with a second set of machine- readable data defining the structure of a chemical entity or moiety of interest using a machine programmed with instructions for evaluating the ability of the chemical entity or moiety to associate with the FRAP or FRAP homolog protein or portion or complex thereof and /or the location and/or orientation of such association.
- Such methods provide insight into the location, orientation and energetics of association of protein surfaces with such chemical entities.
- Chemical entities that are capable of mimicking rapamycin's ability to associate with FRAP or a FRAP homolog should share part or all of rapamycin's pharmacologic activities, e.g. immunosuppressive activity, but may be designed for more convenient or economical preparation, improved pharmacokinetics, reduced side effects, etc. Such chemical entities therefore include potential drug candidates.
- the three dimensional structure defined by the data may be displayed in a graphical format permitting visual inspection of the structure, as well as visual inspection of the association of the protein component(s) with rapamycin or other chemical entities.
- more quantitative or computational methods may be used.
- one method of this invention for evaluating the ability of a chemical entity to associate with any of the molecules or molecular complexes set forth herein comprises the steps of: (a) employing computational means to perform a fitting operation between the chemical entity and a binding pocket or other surface feature of the molecule or molecular complex; and (b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket.
- This invention further provides for the use of the structural coordinates of a crystalline composition of this invention, or portions thereof, to identify reactive amino acids, such as cysteine residues, within the three-dimensional structure, preferably within or adjacent to a ligand binding site; to generate and visualize a molecular surface, such as a water-accessible surface or a surface comprising the space-filling van der Waals surface of all atoms; to calculate and visualize the size and shape of surface features of the protein or complex, e.g., ligand binding pockets; to locate potential H-bond donors and acceptors within the three-dimensional structure, preferably within or adjacent to a ligand binding site; to calculate regions of hydrophobicity and hydrophilicity within the three-dimensional structure, preferably within or adjacent to a ligand binding site; and to calculate and visualize regions on or adjacent to the protein surface of favorable interaction energies with respect to selected functional groups of interest (e.g.
- reactive amino acids e.g., cysteine
- complementary characteristics e.g., size, shape, charge, hydrophobicity /hydrophilicity, ability to participate in hydrogen bonding, etc.
- the structural coordinates of the FRAP or FRAP homolog protein, or portion or complex thereof are entered in machine readable form into a machine programmed with instructions for carrying out the desired operation and containing any necessary additional data, e.g. data defining structural and /or functional characteristics of a potential ligand or moiety thereof, defining molecular characteristics of the various amino acids, etc.
- One method of this invention provides for selecting from a database of chemical structures a compound capable of binding to FRAP or a FRAP homolog.
- the method starts with structural coordinates of a crystalline composition of the invention, e.g., coordinates defining the three dimensional structure of FRAP or a FRAP homolog or a portion thereof or a complex thereof. Points associated with that three dimensional structure are characterized with respect to the favorability of interactions with one or more functional groups.
- a database of chemical structures is then searched for candidate compounds containing one or more functional groups disposed for favorable interaction with the protein based on the prior characterization. Compounds having structures which best fit the points of favorable interaction with the three dimensional structure are thus identified.
- a first set of machine-readable data defining the 3D structure of a FRAP or FRAP homolog protein, or a portion or protein-ligand complex thereof is combined with a second set of machine readable data defining one or more moieties or functional groups of interest, using a machine programmed with instructions for identifying preferred locations for favorable interaction between the functional group(s) and atoms of the protein.
- a third set of data i.e. data defining the location(s) of favorable interaction between protein and functional group(s) is so generated. That third set of data is then combined with a fourth set of data defining the 3D structures of one or more chemical entities using a machine programmed with instructions for identifying chemical entities containing functional groups so disposed as to best fit the locations of their respective favorable interaction with the protein.
- Compounds having the structures selected or designed by any of the foregoing means may be tested for their ability to bind to FRAP or a FRAP homolog, inhibit the binding of FRAP or a FRAP homolog to a natural or non-natural ligand therefor (e.g. FKBP12-rapamycin, in the case of FRAP), and/or inhibit a biological function mediated by FRAP or the FRAP homolog.
- a natural or non-natural ligand therefor e.g. FKBP12-rapamycin, in the case of FRAP
- This invention also permits methods for designing a compound capable of binding to a FRAP or FRAP homolog based on the three dimensional structure of bound rapamycin.
- One such method involves graphically displaying a three-dimensional representation based on coordinates defining the three-dimensional structure of a FRAP or FRAP homolog protein or a portion thereof complexed with a ligand such as the FKBP12:rapamycin complex. Interactions between portions of ligand and protein are characterized in order to identify candidate moieties of the ligand for replacement.
- One or more portions of the ligand which interact with the protein may be replaced with substitute moieties selected from a knowledge base of one or more candidate substitute moieties, and /or moieties may be added to the ligand to permit additional interactions with the protein.
- substitute moieties selected from a knowledge base of one or more candidate substitute moieties, and /or moieties may be added to the ligand to permit additional interactions with the protein.
- FIG. 1 depicts a computer system.
- FIG. 2 depicts storage media of this invention.
- FIG. 3 depicts a ribbon diagram of the three dimensional structure of the FKBP12:rapamycin:FRB domain complex, as defined by the coordinates of Appendix I.
- FRAP is one of a number of PIK-related kinase family members that contain an FRB domain.
- PIK-related kinase family members share regions of homology including lipid kinase homologous regions, kinase domains and, in at least a number of cases, FRB domains.
- the presence and boundaries of homologous regions in a protein sequence can be identified by using a computer alignment program that identifies amino acid sequence homology to a known sequence or domain.
- the FRB domain (amino acids 2015 - 2114) of FRAP may be used for such analysis, but FRB domains from other proteins such as RAPT or TORI or TOR2 can be used as well.
- Needleman-Wunch alignment See e.g., "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins.” Needlman, S.B.; Wunch, CD. /. Mol. Biol. 1970, 48 , 443-453.
- FRAP FRB domain was expressed as a glutathione-S-transferase (GST) fusion protein.
- GST glutathione-S-transferase
- the cDNA encoding residues 2015 - 2114 from human FRAP was cloned into a pGEX vector and expressed in E coli, the resulting fusion protein was recovered and cleaved to yield the FRB protein which was then purified, all as described in detail below.
- FKBP12 protein was similarly obtained using a cDNA encoding residues 1 - 107 from human FKBP12 (Standaert et al, 1990, Nature 246.: 671-674..
- FRB proteins containing an FRB domain may also be used, including larger FRAP fragments containing the FRB and flanking peptide sequence, including up to the entire FRAP protein.
- FRB proteins can be prepared by analogous means containing homologous FRB regions from other proteins, including RAFT, TORI, TOR2 or other members of the PIK-related kinase family.
- other expression systems may be readily employed., including , e.g., materials and methods for expression in E. coli using T7, maltose-binding protein fusion (MBP), with epitope tags (His6, HA, myc, Flag) included or cleaved off.
- Baculoviral expression may be used, e.g. using pVL1393 or derivatives, for tFRB domain, fused (or not) to epitope tag or fusion partner such as GST.
- Conventional materials and methods for expression in mammalian, yeast or other cells may also be used.
- Rapamycin may be prepared by known methods or may be obtained from commercial sources. Rapamycin analogs such as disclosed, e.g., in Luengo et al, 1995, Chemistry & Biology 2(7):471-481, may be used in place of rapamycin, in forming complexes of this invention.
- the ternary complex of FKBP12-rapamycin-FRB has overall dimensions of 60 A x 45 A x 35 A with the rapamycin sandwiched between FKBP12 and FRB.
- the FKBP12 structure is basically the same as in previously reported binary structures, with a five stranded anti parallel ⁇ -sheet and a short ⁇ -helix. This binary structure was originally determined in the FKBP12-FK506 complex and later in the FKBP12-rapamycin complex (Van Duyne et al, 1993).
- the four helix bundle of FRB does not wrap around the effector site of FKBP12-rapamycin; it just touches the effector (i.e., FRB-binding) interface of the binary complex with few protein- protein interactions. All of the interactions between rapamycin and FRB are hydrophobic interactions, and protein-protein interactions between FKBP12 and FRB are limited to the 80s loop and one side chain of the 40s loop of FKBP12 (Table 2).
- the loop-loop interaction between 80s loop (FKBP12) and the ⁇ l- ⁇ 2 loop (FRB) and the loop-helix interaction between 40s loop (FKBP12) and helix ⁇ 4 are the main protein-protein interactions in this ternary complex and thus contribute all of the protein- protein binding force forming the ternary complex.
- the FRB domain of the FRAP forms a typical four helix bundle, which is one of the most common structural motifs in globular proteins.
- the overall dimensions of this domain are 45 A x 30 A x 30 A.
- All four helices (termed ⁇ l- ⁇ 4) are connected with short underhand loops.
- the longest helix cc3 (residues 2065-2091) has a bend at residue 2074 of 59°. Except for a small bent part of ⁇ 3 (residues 1065-2073), all four helices have similar lengths (16-19 residues, about 30 A in length).
- the ⁇ 2 helix also has a small bend around residues Glu2049, Val2050 and Leu2051 to form a 3 ⁇ o-helical turn rather than a normal ⁇ -helix.
- the angle between ⁇ l and ct2 is 22° and the angle between ⁇ 3 and ⁇ 4 is 20°. The angles between these pairs are in the range of 40-60°, which indicates that this four helix bundle is close to the 'X' type interhelical
- FKBP12 in the ternary complex is basically the same as that in the binary complex of FKBP12-rapamycin or FKBP12-FK506.
- the protein fold and the architecture of the secondary structure are exactly the same as in the binary complex, and the interaction with rapamycin is also the same as that of the binary complex.
- FKBP12-rapamycin binding to FRAP was calculated by the rigid-body fitting on the main chain atoms in the FKBP12 using QUANTA.
- the overlay of FKBP12-FK506 to the ternary complex clearly confirmed the fact that FKBP12-FK506 complex can't bind FRAP as FK506's effector region does not extend enough.
- the protein-protein interactions by themselves between FKBP12 and FRB are not enough for the formation of a binary complex; rapamycin is essential to mediate the interaction of the two proteins. FKBP12-rapamycin binding to FRAP
- rapamycin-FKBP12 interactions employ five hydrogen bonds which are the same found in the binary complex of FKBP12-rapamycin, to govern this interaction. Rapamycin is surrounded by five conserved aromatic residues in FKBP12, which makes the binding pocket for the rapamycin a complete aromatic pocket' along with six aromatic residues in FRB domain.
- the other site is Thr2098 which has a close contact with C50 of rapamycin.
- C50 of the rapamycin is at the end of C16 methoxy group, which has been a key target for substituted analogs. All of the hydrophobic interactions between rapamycin and FRB including Ser2035 and Thr2098 can be considered as the main force contributing to complete ternary complex.
- C16 has been the main site for substitution in published structure-activity studies (Luengo et al, 1995). The studies of C16 analogs of rapamycin showed that the bulky group substitutions on this position have lower affinity for the FKBP12 binding and lower activity. However some analogs with different stereochemistry or different groups showed retained activity and affinity to FKBP12. Such C-16 substituted analogs could be of therapeutic use.
- This invention encompasses crystalline compositions containing FRAP or a FRAP homolog protein or portion thereof having a region characterized by structural coordinates of the FRB domain set forth in Appendix I, or by coordinates having a root mean square deviation therefrom of less than about 1.5 A, preferably less than about 1 A, and even more preferably less than about 0.5 A, with respect to backbone atoms of amino acid residues listed there.
- various computational analyses may be used to determine the degree of similarity between the three dimensional structure of a given protein (or a portion or complex thereof) and FRAP or a FRAP homolog protein or portion (e.g. the FRB domain) or complex thereof such as are described herein.
- analyses may be carried out with commercially available software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, MA) version 3.3, and as described in the accompanying User's Guide, Volume 3 pgs. 134 - 135.
- the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
- the procedure used in Molecular Similarity to compare structures is divided into four steps: (1) load the structures to be compared; (2) define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results.
- Each structure is identified by a name.
- One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures).
- atom equivalency within QUANTA is defined by user input, for the purpose of this invention we define equivalent atoms as protein backbone atoms (N, C ⁇ , C and O) for all conserved residues between the two structures being compared and consider only rigid fitting operations.
- the working structure is translated and rotated to obtain an optimum fit with the target structure.
- the fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
- root mean square deviation means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object.
- the "root mean square deviation” defines the variation in the backbone of a protein from the backbone of a protein of this invention, such as the FRB of FRAP, as defined by the structural coordinates of Appendix I and described herein.
- least squares refers to a method based on the principle that the best estimate of a value is that in which the sum of the squares of the deviations of observed values is a minimum.
- the structural coordinates generated for a crystalline substance of this invention e.g. the structural coordinates of the FRB of FRAP set forth in Appendix I
- a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, e.g. a computer loaded with one or more programs of the sort identified above, is capable of displaying a graphical three- dimensional representation of any of the molecules or molecular complexes described herein.
- Machine-readable storage media comprising a data storage material include conventional computer hard drives, floppy disks, DAT tape, CD-ROM, and other magnetic, magneto- optical, optical, floptical and other media which may be adapted for use with a computer.
- a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structural coordinates of a complex, FRB-containing protein component thereof, or portion thereof, comprising structural coordinates of an FRB domain such as the FRAP FRB coordinates set forth in our attached Appendix I ⁇ a root mean square deviation from the conserved backbone atoms of the amino acids thereof of not more than 1.5 A.
- An illustrative embodiment of this aspect of the invention is a conventional 3.5" diskette, DAT tape or hard drive encoded with a data set, preferably in PDB format, comprising the coordinates of our Appendix I.
- the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in Appendix I (or again, a derivative thereof), and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data.
- FIG. 1 illustrates one version of these embodiments.
- the depicted system includes a computer A comprising a central processing unit (“CPU"), a working memory which may be, e.g., RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines (IP), and one or more output lines (OP), all of which are interconnected by a conventional bidirectional system bus.
- CPU central processing unit
- working memory which may be, e.g., RAM (random-access memory) or “core” memory
- mass storage memory such as one or more disk drives or CD-ROM drives
- CRT cathode-ray tube
- IP input lines
- OP output lines
- Input hardware B coupled to computer A by input lines, may be implemented in a variety of ways.
- Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line L.
- the input hardware may comprise CD-ROM drives or disk drives D.
- a keyboard may also be used as an input device.
- Output hardware coupled to computer A by output lines, may similarly be implemented by conventional devices.
- output hardware may include a CRT display terminal for displaying a graphical representation of a protein of this invention (or portion thereof) using a program such as QUANTA as described herein.
- Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
- the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage and accesses to and from working memory, and determines the sequence of data processing steps.
- a number of programs may be used to process the machine-readable data of this invention. Examples of such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system of FIG. 1 are included as appropriate throughout the following description of the data storage medium.
- FIG. 2A shows a cross section of a magnetic data storage medium 100 which can be encoded with a machine-readable data that can be carried out by a system such as a system of FIG. 1.
- Medium 100 can be a conventional floppy diskette or hard disk, having a suitable substrate 101, which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides, containing magnetic domains (not visible) whose polarity or orientation can be altered magnetically.
- Medium 100 may also have an opening (not shown) for receiving the spindle of a disk drive or other data storage device 24.
- the magnetic domains of coating 102 of medium 100 are polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as a system of FIG. 1.
- FIG. 2B shows a cross section of an optically-readable data storage medium 110 which also can be encoded with such machine-readable data, or set of instructions, which can be carried out by a system such as a system of FIG. 1.
- Medium 110 can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable.
- Medium 100 preferably has a suitable substrate 111, which may be conventional, and a suitable coating 112, which may be conventional, usually of one side of substrate 111. In the case of CD-ROM, coating 112 is reflective and is impressed with a plurality of pits
- a protective coating 114 which preferably is substantially transparent, is provided on top of coating 112.
- coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown).
- the orientation of the domains can be read by measuring the polarization of laser light reflected from coating 112.
- the arrangement of the domains encodes the data as described above.
- Structure-based approaches include de Novo molecular design, computer-aided optimization of lead molecules, and computer-based selection of candidate drug structures based on structural criteria.
- Rapamycin mimetics may be developed from the bound conformation of rapamycin by design, by searching databases for replacements of one or more structural segments of rapamycin, or by enhancement of existing ligand-protein interactions (i.e., by replacing a component moiety of a ligand with a substitute moiety capable of greater interaction with the target protein, whether through accessible protein contact points or by extrusion of otherwise sequestered waters).
- Knowledge of the bound conformation of a ligand can suggest avenues for conformational restriction and replacement of atoms and/or bonds of rapamycin.
- a less biased approach involves computer algorithms for searching databases of three dimensional structures to identify replacements for one or more portions of the ligand.
- Compounds designed, selected and /or optimized by methods described above may be evaluated for binding activity with respect to proteins containing one or more FRB domains using various approaches, a number of which are well known in the art. For instance, compounds may be evaluated for activity as competitive inhibitors of the binding of a natural ligand for the FRB, e.g. FKBP12:rapamycin in the case of the FRAP FRB. Competitive inhibition may be determined using any of the numerous available technologies known in the art.
- Such compounds may be further evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the interaction of interest using a suitable cell-based assay or an animal model.
- Cell-based assays and animal models suitable for evaluating inhibitory actvity of a compound with respect to a wide variety of cellular and other biological events are known in the art. New assays and models are regularly developed and reported in the scientific literature. For example, compounds which mimic the binding of rapamycin or FKBP12:rapamycin with respect to FRAP may be evaluated for biological activity in the mouse spelocyte mitogenesis assay or the high-flux yeast-based assay of Luengo et al, supra.
- a battery of in vivo models may be used to profile the breadth of the compound's immunosuppressive (or other) activity and compare the profile to those of positive controls such as rapamycin itself. Comparisons may also be made to other currently accepted immunosuppressive compounds, e.g. cyclophosphamide, and leflunomide.
- Initial in vivo screening models include: Delayed type hypersensitivity testing, Allogeneic skin transplantation, and Popliteal lymph node hyperplasia. Compounds demonstrating optimal profiles in the above models are advanced into more sophisticated models designed to confirm immunosuppressive activity in specific therapeutic areas including: Rheumatoid arthritis, Transplantation, Graft vs. host disease, and Asthma.
- compounds may be evaluated in relevant conventional in vitro and in vivo assays for inhibition of the initiation, maintenance or spread of cancerous growth. See e.g., Ishii et al, J. Antibiot. XLI 1877-1878 (1989) (in vitro evaluation of cytotoxic /antitumor activity); Sun et al, US Patent 5,206,249 (issued 27 April 1993)(m vitro evaluation of growth inhibitory activity on cultured leukemia cells); and Sun et al, supra (xenograft models using various human tumor cell lines xenografted into mice, as well as various transgenic animal models).
- Single and multiple (e.g., 5 to 7 days) dose investigative toxicology studies are typically performed in the efficacy test species using the intended route of administration for the efficacy study. These investigative toxicology studies are performed to identify maximum tolerated dose, subjective bioavailability from the intraperitoneal or oral routes of administration , and estimation of an initial safety margin. Initial bioavailability and pharmacokinetics (blood clearance) of the compounds may be determined, with standard cold or radioactive assay methods, to assist in defining appropriate dosing regimens for the compounds in the animal models.
- compositions and Uses of rapamycin mimetics and other FRAP-binding compounds are provided.
- Compounds which bind to an FRB domain may be used as biological reagents in binding assays as described herein for functional classification of members of the PIK-related kinase family, particularly newly discovered proteins, based on ligand specificity.
- compounds identified as described above can be used for their immunosuppressive or other pharmacologic activity in place of rapamycin.
- a compound selected or identified in accordance with this invention can be formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier and /or other excipient(s) using conventional materials and means.
- a composition can be administered as an immunosuppresant, for example, to an animal, either human or non-human. Administration of such composition may be by any conventional route (parenteral, oral, inhalation, and the like) using appropriate formulations as are well known in this art.
- the compound can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration.
- compositions and methods for treatment or prevention of various diseases and disorders in a mammal in need thereof.
- Mammals include rodents such as mice, rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, non-human primates and humans.
- the preferred method of such treatment or prevention is by administering to a mammal an effective amount of the compound to prevent, alleviate or cure said disease or disorder.
- effective amounts can be readily determined by evaluating the compounds of this invention in conventional assays well-known in the art, including assays described herein.
- the invention provides methods of treating, preventing and /or alleviating the symptoms and /or severity of an untoward immune response or other disease or disorder referred to above by administration to a subject of a in an amount effective therefor.
- the subject will be an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
- Various delivery systems are known and can be used to administer the compound, e.g., encapsulation in liposomes, microparticles, microcapsules, etc.
- One mode of delivery of interest is via pulmonary administration, as detailed more fully infra.
- Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes.
- the compound may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
- preferred routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.
- compositions comprise a therapeutically (or prophylactically) effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
- a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
- the carrier and composition can be sterile. The formulation should suit the mode of administration.
- the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
- the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
- the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
- compositions for intravenous adrrtinistration are solutions in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- composition is to be administered by infusion
- it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- Administration to an individual of an effective amount of the compound can also be accomplished topically by administering the compound(s) directly to the affected area of the skin of the individual.
- the compound is administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
- a topical carrier include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
- Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
- the compound may be disposed within devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
- the effective dose of the compound will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered in single or multiple doses.
- the compound may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
- the amount of the compound which will be effective in the treatment or prevention of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
- in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
- the precise dosage level of the compound, as the active component(s), should be determined as in the case of all pharmaceutical treatments, by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
- the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
- the compound is administered by pulmonary administration, e.g. via aerosolization.
- This route of administration may be particularly useful for treatment or prophylaxis of bronchial or pulmonary infection or tumors.
- Pulmonary administration can be accomplished, for example, using any of various delivery devices known in the art (see e.g., Newman, S.P., 1984, in Aerosols and the Lung , Clarke and Davia (eds.), Butterworths, London, England, pp. 197-224; PCT Publication No. WO 92/16192 dated October 1, 1992; PCT Publication No. WO 91/08760 dated June 27, 1991; NTIS Patent Application 7-504-047 filed April 3, 1990 by Roosdorp and Crystal), including but not limited to nebulizers, metered dose inhalers, and powder inhalers.
- Ultra vent nebulizer (Mallinckrodt, Inc., St. Louis, Missouri); Acorn II nebulizer (Marquest Medical Products, Englewood, Colorado), Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina); Spinhaler powder inhaler (Fisons Corp., Bedford, Massachusetts) or Turbohaler (Astra).
- Ultra vent nebulizer Meltrodt, Inc., St. Louis, Missouri
- Acorn II nebulizer Marquest Medical Products, Englewood, Colorado
- Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina)
- Spinhaler powder inhaler (Fisons Corp., Bedford, Massachusetts) or Turbohaler (Astra).
- Such devices typically entail the use of formulations suitable for dispensing from such a device, in which a propellant material may be present.
- Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis," Consensus Conference, Clinical Outcomes for Evaluation of New CF Therapies, Rockville, Maryland, December 10-11, 1992, Cystic Fibrosis Foundation).
- a nebulizer may be used to produce aerosol particles, or any of various physiologically acceptable inert gases may be used as an aerosolizing agent.
- Other components such as physiologically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, and diluents may also be included. This invention is not to be limited in scope by the specific embodiments described herein.
- the samples containing FKBP12 or FRAP12 were concentrated to -10 mL in a 50 mL stir-cell ultraconcentrator (Amicon) with a 3-kDa cutoff filter, and fractionated on a Sephacryl S-100 column (2.5 cm x 85 cm) equilibrated in 10 mM phosphate buffer (pH 7.4) containing 136 mM NaCl, 3 mM KC1, 1 mM DTT. Fractions containing pure FKBP12 or FRAP12 (>95% purity judged by SDS-PAGE) were combined and concentrated to -10 mg/mL using a stir-cell ultraconcentrator. The concentrated samples were stored in the same phosphate buffer at 4 °C.
- All data sets were collected at room temperature on a San Diego multiwire area detector system mounted on a Rigaku RU-200 rotating anode X-ray source operating at 50 kV and 150mA.
- the detector was positioned at a 20-value of -30° with a 544 mm detector-crystal distance for the high resolution data and 12° with a 506 mm detector-crystal distance for the low resolution data.
- the data collection was performed using an co-scan with an increment of 0.10° for each frame and 40 second exposure time per frame.
- Hg-derivative crystal data were collected under the same conditions.
- the measured intensity data were processed using SCALEPACK (Otwinski et al, 1992) giving 6920 unique reflections out of 43447 measured reflections to 2.7 A resolution (98.5% data coverage) with R S y m of 7.1%.
- the number of unique reflection was 6884 out of 42681 measured reflections to 2.7 A (98.0% data coverage), with R S y m of 7.1%.
- the crystal structure of the ternary complex was solved using the molecular replacement (MR) method combined with the single isomorphous replacement with anomalous scattering (SIRAS) method.
- Initial phases were obtained from the molecular replacement search using the FKBP12-rapamycin complex structure as a search model.
- Rigid body refinement resulted in an R factor of 0.449 (10-2.7 A).
- the FKBP12-rapamycin part was well defined in the initial electron density map; only minor changes in the backbone of 30s loop and some side chains were enough to fit the model of FKBP12-rapamycin structure to this electron density map.
- For the FRB domain part most of a polyalanine chain could be traced for the helix regions in the initial map. After several cycles of the positional refinement using X-PLOR, loop regions could be traced and the amino acid sequence could be assigned.
- CHAIN (Sack, 1988) was used for the model fitting and building the ternary complex.
- the current structure includes 202 amino acids (107 for FKBP12 and 95 for FRB domain), one rapamycin, and 23 water molecules.
- the final R factor is 19.3% with an Rfree of 29.9%.
- the free R-factor is calculated with 10% of the data that were selected at the beginning of the analysis. Crystallographic statistics are summarized in Table 1.
- the final coordinates have good geometry and r.m.s. deviations from the ideality are 0.008 A for bond lengths and 1.5° for bond angles.
- the program PROCHECK (Laskowski, 1993)
- the current 2.7 A resolution structure shows that the main-chain and side- chain geometrical parameters are better than expected at this resolution with an overall G- factor of 0.0.
- Ramachandran plots of ⁇ , ⁇ , angles showed that 86% of the nonglycine and nonproline residues are in energetically most favored regions.
- the average temperature factors for total atoms and main-chain atoms are 17.0 and 14.7 A ⁇ respectively.
- the r.m.s. variation in the B-factor of bonded atoms is 2.5 A ⁇ .
- the Luzzati plot (Luzzati, 1952) indicates that the average coordinate error of this complex structure is between 0.25 and 0.30 A.
- Protein Databank format in Appendix I , below. Such data may be transferred to any desired medium, and formatted as desired, for the practitioner's computer.
- This invention encompasses those coordinates as well as any translation or rotation or the like thereof which maintains the internal coordinates, i.e., which maintains their intrinsic, internal relationship.
- the coordinates may be subjected to other transformations including, e.g. molecular mechanics calculations such as dynamic simulation, minimization, etc.
- This invention further encompasses the use of coordinates of the FRB of FRAP, of the ternary complex, or of the corresponding region of FRAP homologs, and in particular, the coordinates set forth in Appendix I, in conducting such transformations (or more extensive transformations such as the generation of alternative conformations), as well as the products of such transformations (i.e., derivatives of the coordinates).
- Compounds which bind to the FRB of FRAP may be evaluated using materials and methods useful for testing the biological or pharmacological activity of rapamycin analogs. See e.g. Luengo et al, 1995. In addition, the following animal models may be used for further evaluation of such compounds:
- sensitizing chemicals such as dinitroflourobenzene or oxazalone. Seven days later the ears of sensitized mice are painted (challenge) with a lower concentration of the compound.
- Antigen processing and presentation, T lymphocyte activation, leukocyte infiltration, humoral mediator release, increased microvascular permeability, and plasma exudation all result from challenge of sensitized mice and lead to edema formation. Edema presents as a two- to three- fold increase in ear thickness within twenty-four hours.
- test compounds or standards can be applied (topical or parenteral) at various times before or after the sensitization or challenge phases. Increased ear thickness is prevented by several compounds including immunosuppressive agents and steroids. This model is a primary model for contact dermatitis.
- Paw pads are injected with one of these agents. Paws increase in volume, and measurements are made between 20 and 30 days later.
- the ability of test compounds to prevent the induction of paw swelling is tested with daily treatment for 12 consecutive days following the injection of inducing agent.
- the ability for the test compounds to reverse the progression of the paw swelling is tested by administration of the compound for 12 consecutive days beginning on the twelfth day following the injection of inducing agent.
- Paw swelling measurements are made by water displacement plethysmography. Histology is also an appropriate endpoint for these studies.
- the MRL/lpr-mouse model described above, is required for the rheumatoid arthritis indication.
- This model is a spontaneous autoimmune model that develops rheumatoid arthritis resembling the human condition, including the presence of circulating rheumatoid factor, pannus formation, and bone and cartilage erosion.
- SYSTEMIC LUPUS ERYTHEMATOSUS SYSTEMIC LUPUS ERYTHEMATOSUS
- Systemic lupus erythematosus is another autoimmune disease with several animal models.
- Several murine strains develop spontaneous SLE.
- One such strain is MRL/lpr-mice.
- These mice over time (20 to 30 weeks) develop auto-antibodies against dsDNA, nuclear antigens, and renal basement membrane. This leads to complement fixation and immune complex formation. Damage to the kidney becomes apparent with the onset of proteinuria.
- Many of the other physiologic, hematologic, and immunologic aberrations described below for the CGVHD model are present.
- Immunosuppressive compounds such as cyclosporin, cyclophosphamide, and leflunomide can prevent and reverse the course of disease in this model.
- these mice also develop pathologies akin to rheumatoid arthritis.
- the murine chronic graft versus host disease model (CGVHD, described below) is a model of SLE that contains many of the clinical features of SLE. Activity in this model has been shown to be predictive of activity in the more clinically relevant SLE models. (f) TRANSPLANTATION
- Allograft transplantation is often used as an initial test of immunosuppressive activity. While this model is useful as a screen, it may be supplemented with assays based on animal transplant models involving transplantation of internal organ (heart, liver, kidney, bone marrow) with use of "clinically acceptable" physiologic endpoints to assess graft survival. Efficacy of test compounds in only a very limited number of these rodent models is required. Following observation of activity in a rodent model, the test compounds are typically tested in further animal models (e.g., canine, porcine or non-human primate). Active compounds decrease acute and chronic rejection and prolong transplant survival. (g) GRAFT VS.
- HOST DISEASE Chronic GVHD can be used to model CD4 + -dependent humoral immunity. It is induced in BDFi mice (which are progeny of DBA/2 male x C57BL/6 female matings) by administering to them isolated spleen:lymph node cells from DBA/2 mice. This results in: a) disregulation and stimulation of CD4 + T lymphocyte (Lyl + ; murine marker) activity due to incompatibilities at MHC II molecules, and b) abnormal T-B lymphocyte cooperation. The resulting pathological state, in many ways, mimics systemic lupus erythematosus (SLE).
- SLE systemic lupus erythematosus
- measurable endpoints develop within 14 days; including, circulating anti-host IgG and IgE antibodies, altered T and B lymphocyte proliferation activity measured in vitro, complement utilization, hemagglutination, slow progressive wasting, dermal aberrations, splenomegaly, lymphoid hyperplasia, and proteinuria. Only a few of these endpoints need to be measured. Active compounds are are those which limit T lymphocyte disregulation and abrogate changes in these variables. Many steroids (e.g., prednisolone), cyclosporine, FK-506, cyclophosphamide, and leflunomide are all active in this model and can be used as positive controls.
- steroids e.g., prednisolone
- cyclosporine cyclosporine
- FK-506, cyclophosphamide cyclophosphamide
- leflunomide are all active in this model and can be used as positive controls.
- the acute GVHD model is also produced in BDFj mice.
- isolated spleen:lymph node cells from C57BL/6 mice are administered. This results in disregulation and stimulation of CD8 + T lymphocytes due to incompatibilities in the MHC I molecules. Elevated cytokine levels and donor clonal expansion occurs. Ultimately, donor cytotoxic T lymphocytes and NK cells rapidly reject host tissue and cause relatively rapid death of the recipient.
- AGVHD The progression of AGVHD in this model is assessed by measurement of hematologic abnormalities (including T cell number and type), cytokine elevations (TNF, IL-1, IL-2, and/or IL-4), low body weight, hypo ⁇ globulinemia, circulating hematologic characteristics indicative of aplastic anemia (granulocytopenia, thrombocytopenia), ex vivo NK or CTL activity, and host survival.
- Active compounds are those which abrogate changes in the variables, and prolong survival over 4 to 6 weeks.
- Asthma offers another opportunity for safe immunosuppressive therapy.
- Atopic asthmatics have antibody mediated hypersensitivity and the often occurring late phase reaction is likened to a DTH response.
- Asthma has only recently been defined as an inflammatory disease (1992). Since then, several publications from prominent asthmatologists demonstrate the presence of activated CD4 + and CD8 + T lymphocytes in bronchoalveolar lavage fluid and blood of atopic asthmatics. The ratios of these cells changes in asthmatic conditions.
- T cell associated cytokines IL-1, IL-2, IL-4, IL-5, and TNF
- Inflammatory events in asthma are now considered to be T lymphocyte driven.
- Initial clinical trials with inhaled cyclosporin suggest that local immunosuppression can ameliorate airway hyperreactivity - the underlying defect in asthma.
- the guinea pig model of antigen-induced pulmonary aberrations is used as a model for asthma. These animals are actively sensitized to ovalbumin to generate high circulating titers of anti-ovalbumin antibody with seroconversion to the IgE class, as is the case with atopic asthmatics. Aerosol challenge of sensitized guinea pigs results in measurable eosinophil rich pulmonary infiltrates (approximately a 16-fold increase in eosinophils), pulmonary edema, and mucous plugging of the small airways; all culminating in the expression of the underlying defect in asthma- airway hyperreactivity (approximately a 3 to 4-fold increase in reactivity). Acute bronchoconstriction is obviously present and points the aforementioned presence of the pathophysiologic sequelae. Active compounds are those which lessen or abrogate such symptoms.
- Luengo J. I., Yamashita, D. S., Dunnington, D., Konialian Beck, A., Rozamus, L. W., Yen, H., Bossard, M. J., Levy, M. A., Hand, A., Newman-Tarr, T., Badger, A., Faucette, L., Johnson, R. K., D'Alessio, K., Porter, T., Shu, A. Y., Heys, R., Choi, J., Kongsaeree, P., Clardy, J., and Holt, D. A. Chemistry & Biology 2, 471-481 (1995).
- ATOM 180 CA GLN 20 -0.571 12.655 32.647 1.00 27.89 FKBP
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP96936867A EP0862621A1 (en) | 1995-10-23 | 1996-10-23 | Crystalline frap complex |
JP9516735A JPH11514379A (en) | 1995-10-23 | 1996-10-23 | Crystalline FRAP complex |
AU74684/96A AU7468496A (en) | 1995-10-23 | 1996-10-23 | Crystalline frap complex |
AU66669/00A AU767092B2 (en) | 1995-10-23 | 2000-10-20 | Crystalline FRAP complex |
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US606995P | 1995-10-24 | 1995-10-24 | |
US60/006,069 | 1995-10-24 |
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JP (1) | JPH11514379A (en) |
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Cited By (2)
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WO1999065939A1 (en) * | 1998-06-18 | 1999-12-23 | Curagen Corporation | INTERACTION OF p27(KIP1) WITH FKBP-12 |
WO2000068366A1 (en) * | 1999-05-07 | 2000-11-16 | E.I. Du Pont De Nemours And Company | D1-c-terminal processing protease: methods for three dimensional structural determination and rational inhibitor design |
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US5353236A (en) * | 1992-04-23 | 1994-10-04 | The Board Of Trustees Of The Leland Stanford University | High-resolution crystallographic modelling of a macromolecule |
WO1994025860A1 (en) * | 1993-04-28 | 1994-11-10 | Immunex Corporation | Method and system for protein modeling |
EP0676471A2 (en) * | 1994-03-08 | 1995-10-11 | American Home Products Corporation | Effector proteins of rapamycin |
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1996
- 1996-10-23 AU AU74684/96A patent/AU7468496A/en not_active Abandoned
- 1996-10-23 EP EP96936867A patent/EP0862621A1/en not_active Withdrawn
- 1996-10-23 JP JP9516735A patent/JPH11514379A/en not_active Ceased
- 1996-10-23 WO PCT/US1996/016953 patent/WO1997015659A1/en not_active Application Discontinuation
- 1996-10-23 CA CA 2229426 patent/CA2229426A1/en not_active Abandoned
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US5353236A (en) * | 1992-04-23 | 1994-10-04 | The Board Of Trustees Of The Leland Stanford University | High-resolution crystallographic modelling of a macromolecule |
WO1994025860A1 (en) * | 1993-04-28 | 1994-11-10 | Immunex Corporation | Method and system for protein modeling |
EP0676471A2 (en) * | 1994-03-08 | 1995-10-11 | American Home Products Corporation | Effector proteins of rapamycin |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999065939A1 (en) * | 1998-06-18 | 1999-12-23 | Curagen Corporation | INTERACTION OF p27(KIP1) WITH FKBP-12 |
WO2000068366A1 (en) * | 1999-05-07 | 2000-11-16 | E.I. Du Pont De Nemours And Company | D1-c-terminal processing protease: methods for three dimensional structural determination and rational inhibitor design |
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EP0862621A1 (en) | 1998-09-09 |
JPH11514379A (en) | 1999-12-07 |
AU7468496A (en) | 1997-05-15 |
CA2229426A1 (en) | 1997-05-01 |
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