Current Topics in Medicinal Chemistry - Volume 3, Issue 12, 2003

For proteins that are found in organisms ranging from bacteria to humans the clear delineation of the cellular function of immunophilins has run a tortuous path which in some ways appears little closer to its final destination. (Galat pp 1315-1349) This journey has been marked by many waypoints along the road that at each stage has given rise to a new nomenclature for these ubiquitous proteins. The scientific odyssey began in 1984 with the description of porcine peptidyl-prolyl isomerase (PPIase) an enzyme identified as capable of catalysing the cis-trans isomerization of peptidyl-prolyl bonds which was proposed as a rate-limiting step in protein folding. However, it is only recently that the importance of PPIase enzymes in protein folding in vivo has been demonstrated with the identification of a number of PPIase enzymes as crucial members of steroid receptor complexes. (Ratajczak pp 1351-1361)A second journey for investigators of PPIases began from a seemingly different starting point with the discovery that the macromolecular target for the immunosuppressant natural product cyclosporin A termed cyclophilin A (CsA) was identical to porcine peptidyl-prolyl isomerase. Subsequently a second enzyme known as FKBP12, also possessing PPIase activity was identified as the target for a second immunosuppressant natural product FK506 and this lead to the PPIase enzymes being referred to as immunophilins, a classification for enzymes that contain both PPIase activity and bind natural product immunosuppressant drugs. Both FK506 and CsA potently inhibit the PPIase activity of their respective immunophilin protein partners, however, the initial hope that somehow the PPIase activity of these compounds was involved in the immune response was soon dashed with the development of potent PPIase activity inhibitors that possessed no immunosuppression activity. Remarkably, despite their structural diversity both CsA and FK506 mediate their biological effects through inhibition of the same protein phosphatase calcineurin (CN) and inhibition of PPIase activity was found to be ancillary to the immunosuupressant effects. Recently this story has taken a new twist with the proposal that immunophilin drug-complexes block additional signal transduction pathways in activated ‘T’ cells and that these T-cell specific pathways maybe a significant contributor to the source of the T-cell specific effects of these compounds (Koyasu pp 1363-1373)At this point a third journey which is still progressing began with the realisation that immunophilins co-localise with calcineurin and as such are expressed at very high levels within the brain. Subsequent studies in animal models of nerve damage identified FK506 and to a lesser extent CsA as potent neuroprotective and neuroregenerative agents. However, this trip did not converge on the same destination (calcineurin) as immunosuppression with non-immunosuppressant immunophilin ligands being found to be equally neurotrophic as FK506. Recent studies have suggested that a specific FKBP protein (FKBP52) is largely the target responsible for these effects, with binding of FKBP52 disrupting steroid hormone receptor complexes leading to activation of specific signal transduction pathways. (Gold pp 1375-1383) However even these results are becoming more complicated by the day with the discovery that several immunophilins associate with the steroid receptor including both FKBP and cyclophilin classes and that specific steroid receptors appear to have preferences for specific immunophilins. Additionally recent studies on protein composition within brain lesions indicate that FKBP12 is overexpressed in a range protein aggregation events characterising a range neurodegenerative diseases. (Achim pp 1385-1391)In each of the above journeys a common feature appears to be the ability of immunophilins to facilitate the association of proteins or to stabilise protein-protein complexes. This hypothesis is further supported by recent studies indicate that immunophilins are involved in a range of other protein-protein complexes including calcium induced calcium release channels (Marks pp 1393-1402), interactions with the TGF-β receptor and HIV Gag protein. In many of these cases substantial structural characterisation has been achieved with more than 100 crystal structures for immunophilin and immunophilin-protein complexes having being determined. (Walkinshaw pp 1403-1425). The role of immunophilins in all of these complexes is not clear with a defined function for their PPIase activity generally difficult to establish but in each case ligands targeting the immunophilin either stabilise a protein-protein complex (immunosuppression) or destabilise the complex (calcium channels, steroid receptor). Within this issue we present a collection of reviews examining the breadth of the many functions exhibited by immunophilin / neurophilin enzymes and

The observation by Von Euler and Gaddum in 1931 that an extract of horse intestine and brain causes peripheral vasodilatation and contraction of intestinal muscles set stage for a number of studies which ultimately lead to the determination of the amino acid sequence of the first member of mammalian tachykinins namely substance P in 1971. Since then two additional tachykinins from mammalian source, neurokinin A and neurokinin B, have been identified. All these peptides share the Cterminal pentapeptide FXGLMa and their family name tachykinin refers to the ability to induce fast, immediate contractile responses of smooth muscle preparations. The receptors that substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) bind are referred to as NK1, NK2 and NK3 receptors respectively.Mammalian tachykinins are derived from two precursor-encoding genes. As a consequence of alternative processing of RNA and polypeptide precursors, there exists a tissue-specific difference in the expression of these peptides. Tachykinins are multifunctional peptides and modulate a variety of biological activities through G-protein coupled receptors. In the central nervous system, they contribute to central cardiovascular regulation, to the control of motor activity and the rate of respiration. Their presence in the hypothalamus and in the pituitary gland suggests a role in the control of neurosecretion. These peptides also exhibit a variety of peripheral activities. They are involved in nociceptive and pain transmission and perception. They cause vasodilatation, salivation and both exocrine and endocrine pancreatic secretion. They are implicated in regulation of nerogenic inflammation, gastrointestinal motility and pulmonary function. They are present in immune cells.Because of the involvement of tachykinins in several pathophysiological events considerable effort is invested in the discovery of antagonists that will be useful drugs in the treatment of human diseases. We have attempted to review some of the latest developments in this area. The following three reviews deal with the NK1, NK2 and non-selective NK antagonists. Background information is included when needed to keep the discussion in proper perspective. It is our hope that these timely reviews will be useful to practicing medicinal chemists interested in the modulation of tachykinin activity to meet unmet medical needs.

Information recovered from genome sequencing projects, multiple sequence alignments, structural analyses of PPIase and published records were used in deciphering the biological diversity, functions and targets of four groups of proteins encoded by dissimilar sets of sequences whose spatial representations exhibit peptidylprolyl cis / trans isomerase activity (PPIase). In the human genome there are encoded fifteen proteins whose segments have significant homology with the sequence of 12 kDa protein which is the target of the potent immunosuppressive macrolides FK506 or rapamycin. The 12 kDa archetype of the FK506-binding protein (FKBP), known as FKBP-12a, is an abundant intracellular protein whereas other FKBPs possessing from one to four FK506- like binding domains (FKBDs) have nominal masses varying from 13 to 135 kDa. The human genome contains at least sixteen genes encoding proteins comprising one cyclosporin-A (CsA) binding domain (CLD) called cyclophilins whose nominal masses vary from 17 to 324 kDa and multiple coding segments for small cyclophilins (17-19 kDa) whose transcription levels and functions remain unknown. The third group of PPIases encoded in the genome comprises two proteins (hPin1 and hParv14) where hPin1 is an important PPIase for cell cycle. The A. thaliana, C. elegans, D. melanogaster and S. cerevisiae genomes encode a less diverse spectrum of PPIases whereas the prokaryotic genomes contain from none to three cyclophilins, from none to four genes encoding FKBPs, one distant homologue of the Pin1 protein named parvulin and the fourth group of PPIases known as trigger factors. PPIases are discretely distributed to different cellular compartments and interact with a number of targets that control a range of cellular processes. Analyses of the sequence alignments of the two groups of PPIases, namely cyclophilins and FKBPs from diverse phyla, show that in each group their sequences diverge but the amino acid residues which form the PPIase activity site and macrolide binding cavity remain well conserved in the majority of them which suggests that the spatial structures and functions of each group of PPIases remain conserved.

The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine / threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIasemediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

It is well established that the immunosuppressive effects of cyclosporin A (CsA) and FK506 (also known as tacrolimus) are mediated through binding to their cognate cellular proteins cyclophilin and FKBP (collectively termed immunophilins), respectively. Biochemical analysis had revealed that cyclophilin-CsA and FKBP-FK506 complexes bind to and inactivate Ca2+- dependent serine / threonine phosphatase calcineurin. Since calcineurin regulates nuclear translocation and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factors that is one of essential steps for cytokine gene expression in activated T cells, it is believed that inhibition of calcineurin is a molecular basis of the immunosuppressive properties of CsA and FK506. However, recent studies indicate that both CsA and FK506 can block activation of JNK and p38 signaling pathways during T cell activation. CsA and FK506, thus, have two distinct mechanisms of action; one is the inhibition of the protein phosphatase activity of calcineurin, leading to the blockade of the nuclear translocation of NFAT transcription factors, and the other is the suppression of JNK and p38 activation pathways. It is likely that the presence of two distinct targets in T cell activation makes CsA and FK506 highly potent immunosuppressive drugs. Here we discuss the action of immunophilin-ligand complexes on JNK and p38 activation pathways. We also argue the possibility of immunotherapeutic application targeting at JNK and p38 signaling pathways.

FK506 (tacrolimus), initially developed as an immunosuppressant drug, represents a class of compounds with potential high impact for the treatment of human neurological disorders. While immunosuppression is mediated by the 12-kD FK506-binding-protein (FKBP-12), the neurite elongation activity of FK506 involves FKBP-52 (also known as FKBP-59 or Hsp-56), a component of mature steroid receptor complexes: FKBP-52 binds to Hsp-90, which bind to p23 and the steroid receptor protein to form the complex. The brief review focuses on how three classes of compounds (FK506 derivatives, steroid hormones, and ansamycin anti-cancer drugs, e.g., geldanamycin) increase neurite elongation / nerve regeneration (axonal elongation). A model is presented whereby neurite elongation is elicited by compounds that bind to steroid receptor chaperone proteins (e.g., FKBP-52 and Hsp-90) and thereby disrupt mature steroid receptor complexes (comprising FKBP-52, Hsp-90 and p23 in addition to the steroid receptor binding protein). Disruption of the complex leads to a “gain-of-function” whereby one or more of these steroid receptor chaperone proteins (i.e, FKBP-52, Hsp-90 or p23) activates mitogen-associated protein (MAP) kinase / extracellular signal-regulated kinase (ERK) pathway. Thus, the neurotrophic actions of these distinct classes of compounds can be understood from their ability to bind steroid receptor chaperones, thereby providing a unique receptor-mediated means to activate the ERK pathway. These studies thereby shed new light on the intrinsic mechanism regulating axonal elongation. Furthermore, this mechanism may also underlie calcineurinindependent neuroprotective actions of FK506. We suggest that components of steroid receptor complexes are novel targets for the design of neuroregenerative / neuroprotective drugs.

Immunophilins are receptors for immunosuppressive drugs like cyclosporin A, FK506, rapamycin and their non- immunosuppressive analogs, which are collectively referred to as “immunophilin ligands” (IPL). Cyclosporin A binds to a class of IP called cyclophilins, whereas the receptors for FK506 and rapamycin belong to the family of FK506- binding proteins (FKBP). The latter are designated according to their molecular weight: FKBP12, 25, 52 etc. FKBP levels in the rat brain are up to 50 times higher than in the immune system. FKBP12 is associated with IP3 and ryanodine receptors present on the endoplasmic reticulum and plays a role in stabilizing calcium release. It has also been proposed to be a modulator of the TGFβ receptor activity. Crush injury of facial or sciatic nerves in rat leads to markedly increased FKBP12 levels in the respective nerve nuclei and this increase is related to nerve regeneration. Cyclophilin A protects cells from death following expression of mutant Cu / Zn superoxide dismutase, which is associated with familial amyotrophic lateral sclerosis. Our recent studies show that FKBP12 and FKBP52 are expressed in the human nervous system, especially in the substantia nigra- deep gray matter axis. In neurodegenerative diseases, FKBP12 levels increase in neurons situated in areas of pathology. This IP colocalizes with synaptophysin and α- synuclein, suggesting that it may become a novel marker of pathology. Immunophilins participate in axonal transport, synaptic vesicle assembly and may play a role in neuroprotection against abnormal protein aggregation, suggesting a potential avenue of therapeutic interventions.

The ryanodine receptor (RyR) is the major calcium (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle and is required for excitation-contraction (EC) coupling. The 565 kDa RyR protein forms a tetrameric channel that is part of a macromolecular signaling complex that also includes four FK506 binding proteins (FKBPs). The RyR channel complex is localized on specialized regions of the SR, such that the large RyR cytoplasmic domain is closely opposed to the transverse tubule (T-tubule) of the plasma membrane. RyR channel complexes are organized in regular arrays such that neighboring RyRs are in physical contact with each other. We have shown that physical and functional association between RyR1 or RyR2 channels results in coordinated gating behavior termed coupled gating. Coupled gating requires FKBP12 or FKBP12.6 in the RyR1 or RyR2 macromolecular complexes, respectively. FKBPs are known to stabilize single RyR channel function. Coupled gating describes an additional role for FKBPs in the functional coordination of RyR channel complexes that allows clusters of channels to function as “Ca2+release units” (CRU). In addition, the FKBP-RyR interaction is regulated by PKA phosphorylation. In failing hearts PKA hyperphosphorylation of RyR2 causes depletion of FKBP12.6 from the channel macromolecular complex and may contribute to contractile dysfunction by impairing EC coupling. As FKBPs are potent modulators of RyR channel function, the FKBP-RyR interaction is a focus for determining molecular mechanisms of coupled gating and presents an exciting pharmacologic target for restoration of RyR complex function in diseased states.

This review includes an analysis of available X-ray and NMR structures of both members of the immunophilin family; cyclophilins and the FK-506 binding proteins (FKBPs). Available structures are compared and contrasted to highlight different structural features seen both within and between species. Each immunophilin family has been structurally characterised with a variety of small molecule ligands, principally immunosuppressive drugs and their analogues and an overview of these complexes is also presented. Currently the Protein Data Base contains over 60 entries for cyclophilins and over 40 entries for FKBPs. A number of FKBP related structures are also available including structures of MIP (Macrophage Infectivity Potentiator protein) from Legionella pneumophila and Trypanosoma cruzi and Trigger Factor from Mycoplasma genitalium. For all structures discussed in the review a summary of the available biological data is also presented.

The mammalian tachykinin (TK) peptides and their three neurokinin (NK) receptors represent an effector system with wide-ranging actions on neuronal, airway smooth muscle, mucosal, endothelial, immune, inflammatory and remodeling cell function. Recent clinical and preclinical data suggests pathophysiological relevance for TKs in various diseases including asthma, emesis and depression. The promiscuous TK-NK receptor interactions and incompletely overlapping functions mediated by each NK receptor may indicate added therapeutic benefit of using multiple NK receptor blockade. Consequently, there has been substantial pharmaceutical effort in projects to develop nonpeptide dual and triple NK receptor antagonists. This review identifies the chemical and biological approach used to develop a TK antagonist active at the three NK receptors. Clinical activity has been observed using single and / or dual NK receptor antagonists in asthma, depression / anxiety and, most notably, emesis trials but no compound with mono or multiple NK receptor antagonist activities has cleared all the development and regulatory hurdles to commercialization. Current experience indicates that potent dual and triple NK receptor-selective antagonists possessing appropriate affinity and pharmacokinetic properties can be developed. As an example, the biological and pharmacokinetic profiles of a new representative of this class of agent, SCH 206272, is detailed in the present review. Whether such agents will fulfill researchers' expectations must await further clinical trials.

The study of tachykinin NK1 (substance P) receptor antagonists has emerged as a field of great promise due to accumulating evidence that NK1 antagonists offer possible new treatment options in therapeutic areas ranging from pain, emesis, and pulmonary disorders to depression and anxiety. It is hoped that the unique mechanism of action of these agents, which involves modulation of effects mediated by the interaction of the neuropeptide substance P with it's Gprotein coupled receptor, will provide improvements over existing therapies. For this reason many pharmaceutical companies are engaged in intense research programs with the goal of bringing safe and effective new drugs to the market. To date a wealth of diverse NK1 antagonists have been discovered, several of which have been evaluated in clinical trials. Despite rich structural diversity in this area of medicinal chemistry a number of structural features are commonly shared amongst otherwise unrelated antagonists. This theme and others are covered with the aim of conveying recent successful approaches to the discovery of potent and selective nonpeptide NK1 antagonists. This review focuses mainly on reports appearing in the year 2001 and the first half of 2002.

The search for new NK2 receptor antagonists have resulted in the discovery of several different classes of compounds with promise to have clinical utility. Clearly, the first reported non-peptide NK2 receptor antagonist (SR- 48,968) has inspired a lot of effort in the area, but over the years other approaches have also been fruitful. These include optimisation of hits from random screening and modifying compounds with NK3 receptor antagonistic properties into selective NK2 receptor antagonists. This is also an area where cyclic peptides and derivatives have been extensively examined. So far, no NK2 receptor antagonist has reached the market, but several clinical trials are in progress.