Targets of S100B
1. Introduction
The development of new therapies for patients diagnosed with malignant melanoma is in high need. In this chapter, the design and testing of inhibitors are discussed for S100B, a calcium-binding protein that down-regulates the tumor suppressor p53. Because p53 is wild type in many malignant melanoma patients, the restoration of p53 with S100B inhibitors (SBiXs) represents a new and potentially effective strategy for sensitizing melanoma cells to p53-dependent apoptosis pathways and for targeting this deadly cancer. Such a strategy requires blocking of the S100B-p53 protein-protein interaction (PPI) and involves methods including computer aided drug design (CADD), screening technologies, nuclear magnetic resonance (NMR), X-ray crystallography, and medicinal chemistry approaches. The ultimate goal is to design a highly specific and potent inhibitor of S100B that has clinical value.
2. The S100 protein family
The S100 family of EF-hand calcium-binding proteins has more than 20 members, with the genes encoding these proteins present only in vertebrates [7]. S100 proteins (S100s) are expressed in both a cell type and tissue-specific manner to provide diverse functional roles including calcium homeostasis, cell-cell communication, cell proliferation, differentiation, cytoskeletal dynamics, and cell morphology [7-10]. On the other hand, dysregulation of S100 expression is observed in several types of cancers, including malignant melanoma [7-9]. They are also problematic when elevated in several cognitive disorders including those arising from traumatic brain injuries [12-16]. While S100 proteins themselves have no inherent enzymatic activity, they regulate important biological processes via specific Ca2+-dependent protein-protein interactions [17,18].
The first members of the S100 family were discovered in 1965 in a subcellular fraction from bovine brain tissue and were named based on their solubility in 100% saturated ammonium sulfate. When this protein fraction was examined in detail, two similar, but distinct proteins were discovered and designated S100α and S100β that are now referred to as S100A1 and S100B, respectively [17,19]. As with S100A1 and S100B, other S100s have a similar molecular weight (9-12 kDa), have homologous amino acid sequences (>40%), and typically exist as symmetric homodimers, or as heterodimers, held together by noncovalent interactions as pairs of four-helix bundles [20,21]. Two EF-hand helix-loop-helix calcium-binding structural motifs, first defined using the “E” and “F” helices from the X-ray crystal structure of parvalbumin, are present in each S100 subunit [22]. The N-terminal “
3. S100B structure & interactions with ions
As with most S100 proteins, each 91 amino acid subunit of S100B has four alpha helices arranged into two helix-loop-helix (HLH) calcium-binding motifs connected by the flexible “hinge” region. Helix 1 and 2 make up the S100 EF-hand, while helix 3 and 4 form the canonical EF-hand (Figure 1). Each S100B subunit, therefore, binds two molecules of calcium, though with significantly different affinities [27]. While the canonical EF-hand binds Ca2+ with moderate affinity (
Thus, as with many EF-hand proteins, S100 signaling proteins do not bind Ca2+ with high affinity unless they are bound to their biologically relevant protein target(s) [32-34,37,38]. In other words, in the absence of a bound target, the Ca2+-binding affinity for most S100 proteins is relatively low (i.e. in the μM range [1,17,27,39], but when bound to peptides (i.e. TRTK-12) or full-length targets, the Ca2+-binding affinity can be increased by 5- to 300-fold, respectively [32-34,37,38,40]. This property is physiologically necessary because while there are over 600 EF-hand Ca2+-binding domains within any given cell, Ca2+ homeostasis must be maintained with sufficient free Ca2+ ion concentrations for proper signaling (i.e. 100 to 500 nM). Thus, as a physiological control mechanism, S100s and many other EF-hand proteins do not sequester significant amounts of free Ca2+ unless their functionally relevant molecular target is available [29,34,38]. It is especially important for drug design that we continue to investigate and understand this phenomenon at the molecular level because S100 inhibitor binding must mimic the EF-hand-target complex and allosterically tighten Ca2+ ion binding affinity upon complex formation to be effective inside the cell [35,37]. For S100B, this includes targets such as p53, hdm2, hdm4, Rsk1 and RAGE, among others, which subsequently contributes to a Ca2+-mediated growth response in a cell-specific manner, including those in skin and brain (Table 1).
4. S100B pathology
The protein S100B is found in melanocytes, glial cells, chondrocytes, and adipocytes, exhibiting both intra- and extracellular functionality. The cellular responses elicited by S100B can vary depending on several factors, including concentration (nM or µM), cell type, and cellular location [8,9]. Of particular concern is the role of elevated S100B in melanoma (Figure 2), the most deadly of all skin cancers, notorious for its resistence to chemotherapy and radiation. Clinical studies have established S100B as an effective biomarker for melanoma; however, this is only the case when highly specific S100B antibodies are used [12]. For example, in one study, samples from 412 melanoma patients at varying stages were compared to those diagnosed with non-melanoma skin cancers and inflammatory cutaneous diseases. Using a cutoff value of 0.2 µg/l serum S100B, a positive correlation was observed for patients having S100B levels above the cutoff level and advancement of tumor stage, indicative of a contribution by S100B to micro- and/or macro-metastases [41-43]. Though elevated S100B cannot be used to identify tumor thickness or lympth node status, it is predictive of poor patient prognosis, increased tumor recurrence, and low overall survival [9,41-44]. Subsequent studies reinforce these findings and consistently show elevated levels of S100B to be a sensitive and specific marker of melanoma progression with the ability to detect metastases or relapse at much earlier timepoints. S100B levels can also be used to monitor treatment strategies for rapid identification of whether a particular therapy is promising or for deciding to take an alternative approach [9]. While S100B is a useful prognostic indicator for melanoma, its use as a biomarker for several other cancers with elevated S100B is still under investigation; including colorectal cancer [45-47], several gliomas [48,49], mengiomas [50], non-small cell lung cancer (NSCLC) [51], renal cell carcinoma (RCC) [52], and thyroid carcinoma [53]. In addition, these clinical observations underscore the need to fully understand the role of elevated S100B in cancer, which is ongoing [2-4,54].
Although not considered in detail here, S100B also plays an important role in the brain, and as with cancer, several cognitive disorders show over-expression of S100B in brain tissue and are associated with pathological states including Alzheimer’s disease (AD), Down’s syndrome (DS), and schizophrenia [55-59]. One mechanism for this pathology is that elevated intracellular levels of S100B present in glial cells are excreted and regulate neighboring neuronal cell activity. At low levels, the presence of this extracellular S100B is sufficient to promote neurite extension and growth, while elevated S100B levels are toxic and lead to neuronal cell apoptosis [9,60]. As with skin cancer, the clinical utility of S100B as a marker to identify and characterize neurological diseases and traumas is complicated by overlapping expression of S100B and other S100s in several cell types, its multiple mechanisms of secretion, and its association with more than one neurodegenerative disorder [14]. However, as found for melanoma, lowering S100B levels upon drug delivery is one means used to evaluate drug efficacy for treating schizophrenia [16,61]. Furthermore, the development of S100B inhibitors themselves may be useful for the treatment of these neuropathies, making the identification of such compounds important for advancing efforts towards understanding and treating cancers and cognitive disorders in which S100B levels are at pathologically high levels [62].
|
|
|
Ca2+ Homeostasis | AHNAK* | [26] |
Cell Cycle Regulation | Hdm2, Hdm4 NDR |
[63] [64,65] |
Cytoskeletal Regulation | Caldesmon*
Calponin CapZα GFAP IQGAP1 MARCKS* Src kinase τ-protein* Tubulin |
[66] [67] [68] [69] [70] [71] [26] [72] [73] |
Energy Metabolism | Fructose 1,6 bisphosphate aldolase Phosphoglucomutase |
[74] [75] |
Growth & Survival | p53* | [1,28,76,77] |
5. S100B targets
The ability of S100B to bind a diverse array of protein and enzyme targets is attributable to its broad consensus target-binding sequence [63]. S100B targets include proteins involved in calcium homeostasis, cell-cycle regulation, cytoskeletal regulation, energy metabolism, and growth/survival (Table 1). One common theme among several S100B-target interactions is that they regulate protein phosphorylation [78]. For example, S100B associates with nuclear Dbf2-related (NDR) protein by binding a region distinct from the active site and inducing a conformational change, which stimulates autophosphorylation, and ultimately activates the protein [64]. S100B also regulates phosphorylation by binding to kinase substrates such as those of protein kinase C (PKC) and sterically blocking phosphorylation [76,77,79] (Table 1). This includes the myristoylated alanine-rich C-kinase substrate (MARCKS), τ-protein, and caldesmon to name a few [66,80,81]. One noteable S100B target is the PKC substrate, p53, which is activated by phosphorylation in the C-terminal negative regulatory domain (NRD). In addition to blocking PKC-dependent phosphorylation, the S100B-p53 complex formation shifts the p53 tetramer to dimer to monomer equilibrium towards oligomer dissociation [76,78]. Thus, for p53, when S100B levels are too high, PKC-mediated activation of p53 is inhibited and p53 tetramers are dissociated. Consequently, p53 cannot bind DNA, which affects its transcriptional activity [2,28,76,77,82,83] and inhibits its ability to control cell cycle progression and apoptosis [2-4]. Other S100B targets include the E3 that designates p53 for ubiquitination, Hdm2, and the Hdm2 regulator, Hdm4 [63]. Thus, studies are underway to understand how S100B complexes involving Hdm2/Hdm4 contribute to lowering p53 levels in melanoma. Complicating this is the fact that both of these negative regulators of p53, Hdm2 and S100B, are themselves transcriptionally regulated by p53 [4,63]. This feedback regulatory mechanism results in time-dependent regulation of p53 that depends on having correct levels of all four proteins for proper regulation of cell cycle growth arrest and apoptosis [63]. Since elevated S100B disrupts the maintenance of p53 levels and promotes a cancerous phenotype, the development of small molecule inhibitors designed to target Ca2+-bound S100B has become a high priority. Specifically, investigations are focused on the identification of compounds capable of blocking the Ca2+-dependent S100B-p53 interaction in malignant melanoma (Figure 3). Ideally, administration of such compounds would reactivate p53 in malignant melanoma, as found for siRNAS100B, to induce normal apoptosis pathways and reduce proliferation/survival of the cancer cells [2-4].
6. Targeting the S100B-p53 interaction
Binding of S100B to p53 blocks PKC-dependent phosphorylation, p53 tetramerization, and p53-dependent transcription activation [28,63,76,82,83]. Therefore, efforts to restore wild-type p53 activities in malignant melanoma are underway as part of a drug design strategy [28]. A combination of approaches is being used, including those involving target validation and screening, computer aided drug design, structural biology, medicinal chemistry, and
Screening for S100B inhibitors was initiated using computer-aided drug design methods (CADD) [28,84], and in all steps of identifying and prioritizing “hits” during these and other screens, the pharmacological activity of compounds was evaluated semi-quantitatively, providing an unbiased means of eliminating compounds that do not fulfill specific “drug-like” criteria [84,85]. Compounds identified in screens are also evaluated regarding their potential for absorption, distribution, and metabolism/excretion (ADME) properties [86]. Among many CADD approaches, a recent structure-based technique termed Site Identification by Ligand Competitive Saturation (SILCS) is now used extensively [87-89]. The simultaneous presence of benzene, propane and water in MD simulations of the target protein (ie. S100B) in this fragment-based computational approach identifies potential binding regions for aliphatic moieties, aromatic moieties and hydrogen bond donors and acceptors, while simultaneously allowing for increased flexibility and conformational changes to occur within the drug-binding site [87-89]. In addition, SILCS is very useful for strategically modifying “hits” or “lead compounds” to span a larger area of the protein surface [87,88]. CADD methods such as these are particularly important for blocking protein-protein interactions (PPIs) such as that for the S100B-p53 complex since at least three distinct target binding pockets have been identified on S100B (Figure 5) [27,29,30,37,63,68,90,91]. As a result, the drug pentamidine diisethionate (Pnt), which is referred to as SBi1 (designated SBiX, where ‘X’ is an arbitrary compound number), was identified at a very early stage of the screening process as an effective inhibitor of the S100B-p53 complex [84]. Pnt was approved by the FDA as an antimicrobial agent for the treatment of Pneumocystis carinii pneumonia (PCP), which allowed for repurposing of this drug for
In addition to CADD, biochemical and cellular screening methods are continuously ongoing to identify “hits” that can be considered further as scaffolds for drug development (Figure 4). One sensitive method done
While it is important to show that an S100B-compound complex forms
One of the most important requirements of any drug development program is to obtain physiological data at an early stage in the process to help determine whether a lead compound is effective and/or shows unanticipated toxicities
In the case of modified leads that have ADME properties favorable for systemic administration, concurrent tolerability (MTD) and pharmacokinetic (PK) assays are also conducted. MTD and PK trials are also performed to determine if a lead is suitable for pre-clinical testing or if it requires additional medicinal chemistry optimization and/or further evaluation prior to pre-clinical testing. If the compound is found to be toxic, then it is eliminated from further consideration. Should successful tumor shrinkage be observed in mice treated with the well-tolerated S100B inhibitors, an effort is then put in place to consider phase 1 or 2b human clinical trials.
|
|
|
|
KD | <10 µM | <50 nM | <50 nM |
IC50 in cells | <10 µM | <50 nM | <50 nM |
Off target effects | KD≈IC50 | KD≈IC50 | KD≈IC50 |
Activity in target (-/-) cells | <50% | <20% | <10% |
CYP2D6 Metabolism | Not determined | No | No |
P450 CYP induction | Not determined | <50% at 30 mM | <50% at 30 mM |
Bioavailability | Not determined | Preferred oral | Preferred oral |
Metabolic stability | Not determined | >80% after 1 hour | >80% after 1 hour |
BSA Ligand KD | Not determined | KD> 10 mM | KD> 10 mM |
Specificity | >5:1 | >50:1 | >500:1 |
7. SBiX lead optimization
SBiX leads are typically optimized using structure-based drug design and by examining structure/activity relationships (SAR) using traditional medicinal chemistry approaches. Modified leads are also tested using cellular and
The availability of 3D structures of S100B-SBiX complexes allow for CADD to be used to select compounds from 3D chemical databases with an enhanced potential for binding to S100B [101-105] and/or to engineer compounds de novo via
8. Summary
Ongoing collaborative efforts involving biology, structure determination, CADD and synthetic chemistry have lead to the development of a collection of inhibitors of S100B. These efforts include identification of the FDA approved compound pentamidine, which is currently being evaluated in human clinical trials. In addition, the work has identified several novel chemical scaffolds that are undergoing optimization and have laid the foundation for the application of fragment-based approaches to design additional novel scaffolds. Notably, while the goal of this research is to develop a potent inhibitor of S100B for the treatment of malignant melanoma, we anticipate that the knowledge gained to date will be of utility in designing specific inhibitors of other members of the S100 protein family for the treatment of a range of S100 associated disease states.
Acknowledgments
Support from the NIH (CA107331; to DJW), The Center for Biomolecular Therapeutics (CBT), and the University of Maryland Computer-Aided Drug Design Center is appreciated.References
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