SBSR 197 S2214-1804(17)30048-X 10.1016/j.sbsr.2017.06.001 The Authors Fig. 1 Binding curve of BVNP-0197 to immobilized NGF. Two-site steady-state binding curve of small molecule NGF-inhibitor BVNP-0197 binding to immobilized NGF. X-axis: concentration (muM); Y-axis: percent binding subtracted response (response measured from active flow cell minus response measured from reference flow cell). Samples run in the absence of BSA are represented in black triangles; samples containing 1% BSA are represented in grey circles. Standard error of the mean is shown with error bars (n=3). Fig. 1 Fig. 2 Kinetic rate analysis of BVNP-0197 binding to immobilized NGF. Subtracted sensogram of BVNP-0197 (200muM) outlining the changes in kinetic rate of small molecule binding to immobilized NGF. Time 0-60s represents the association of BVNP-0197 to NGF; at 60s the analyte flow stops. A dissociation period from 60 to 90s is monitored before regeneration of the chip occurs before the next sample is run. Black: BVNP-0197 binding to NGF in the absence of BSA. Grey: BVNP-0197 binding to NGF in the presence of BSA. (A) BVNP-0197 shows a decreased rate of association with the presence of BSA to immobilized NGF. (B) BVNP-0197 shows an increased rate of dissociation with the presence of BSA from immobilized NGF. Fig. 2 Fig. 3 Relationship between binding data from cell-free and cell-based assays. X-axis: IC50measured from cell-based125I-NGF binding (muM); Y-axis: High affinity KDmeasured using cell-free SPR binding to immobilized NGF (muM). SPR analysis was completed in the presence (black triangle) and absence (grey circles) of 1% BSA during binding to immobilized NGF. SPR binding in the presence of BSA was correlated to the cell-based binding measurements (F(1,12)=5.070; p=0.0480; R2=0.3364) (dotted black line). This relationship was not statistically significant when SPR binding was completed in the absence of BSA (solid black line). Fig. 3 Table 1 Binding measurements to NGF in cell-free and cell-based assays. Table 1 Compound KDto NGF-SPR binding (muM) IC50125I-NGF Binding (muM) 0% BSA 1% BSA BVNP-0193 22.4 1.43 0.001 BVNP-0198 5.61 0.45 0.003 BVNP-0183 4.03 0.86 0.013 BVNP-0195 9.61 2.92 0.013 BVNP-0197 21.0 0.43 0.030 BVNP-0191 5.13 4.81 0.033 BVNP-0196 61.0 1.19 0.070 BVNP-0190 4.34 4.23 0.080 BVNP-0182 57.7 1.62 0.530 BVNP-0188 14.0 4.21 1.830 BVNP-0194 16.0 2.37 50.00 Ro 08-2750 36.9 8.88 86.00 Effects of serum albumin on SPR-measured affinity of small molecule inhibitors binding to nerve growth factor Allison E. Kennedy a b allkennedy@nosm.ca Rahul Vohra c John A. Scott d Gregory M. Ross b ? greg.ross@nosm.ca a Laurentian University, Biomolecular Sciences, Sudbury, ON, Canada Laurentian University Biomolecular Sciences Sudbury ON Canada b Northern Ontario School of Medicine, Sudbury, ON, Canada Northern Ontario School of Medicine Sudbury ON Canada c Sussex Research Laboratories Inc., Ottawa, ON, Canada Sussex Research Laboratories Inc. Ottawa ON Canada d Laurentian University, Bharti School of Engineering, Sudbury, ON, Canada Laurentian University Bharti School of Engineering Sudbury ON Canada ? Corresponding author at: Northern Ontario School of Medicine, 935 Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada. Northern Ontario School of Medicine 935 Ramsey Lake Road Sudbury ON P3E 2C6 Canada Abstract The study of the interactions between a drug and plasma serum proteins are necessary in determining pharmacological and toxicological properties for therapeutic development. Small molecule nerve growth factor (NGF) inhibitors have been investigated for their abilities to inhibit NGF binding to TrkA as a potential therapeutic option for the treatment of neuropathic and inflammatory pain. In this study, surface plasmon resonance (SPR) spectroscopy and125I-NGF radioisotope binding assays were carried out to better understand the role of serum albumin (SA) in small molecule binding to NGF. SA has been characterized as a universal drug carrier with up to seven binding domains on its surface to transport drug molecules to target tissues. Here, we use SPR kinetic analysis to analyze the change in specificity of small molecules to immobilized NGF in the presence and absence of SA. In the presence of SA an overall increase in small molecule binding affinity for NGF was observed compared to binding in the absence of SA. Our results suggest a crucial role for SA in the pharmacokinetics of small molecule binding to NGF. This effect will require consideration when developing therapeutic agents. Graphical abstract Image 1 Keywords NGF Drug binding Pain Therapeutic Surface plasmon resonance Pharmacokinetics 1 Introduction Nerve growth factor (NGF) functions not only in prenatal nerve growth, but also has a significant role in pain and immune function in adults[1,2]. During tissue damage, NGF, interleukins and tumour necrosis factor-alpha are secreted by immune cells resulting in painful pathologies[3,4]. NGF in particular leads to hyperalgesia and allodynia by triggering peripheral and central neuronal sensitization by activating the receptor TrkA[5,6]. In such conditions, having an analgesic with the ability to block NGF binding to TrkA and the resulting painful signal would hold great therapeutic advantage. It is estimated that 20-30% of adults suffer from chronic pain, many of whom are not adequately relieved from the painful symptoms with current therapeutic options[7,8]. Targeting NGF-TrkA signalling for the development of novel therapeutic agents has been the focus of several studies. Currently the humanized monoclonal anti-NGF antibody Tanezumab is in Phase III clinical trials for sequestering free NGF for the treatment of arthritic joint pain[9]. Despite being highly selective for NGF, there are many challenges to antibodies as a therapeutic agent including autoimmune responses, delivery challenges and production capacity[10]. Small molecule-based inhibitors have emerged as a possible therapeutic option for inhibiting NGF. Small molecule NGF-inhibitors such as PD 90780[11], ALE 0540[12], Ro 08-2750[13]and PQC-083[14]have demonstrated their efficacy in inhibiting NGFin vitroand in some casesin vivo. Flexible docking experiments suggest that these small molecule inhibitors bind to NGF at the loop I/IV cleft to alter the molecular topology in a manner which alters the binding capacity for the receptor TrkA[15]. More recently, a series of bivalent naphthalimide analogues were screened using surface plasmon resonance (SPR) spectroscopy to identify lead small molecule NGF-inhibitors with nanomolar inhibitory efficiency binding to immobilized TrkA (in press). Serum albumin (SA) is an abundant plasma protein in the circulatory system that functions as the main transport vehicle for fatty acids[16,17]. SA is also capable of transporting a wide variety of drug molecules, which affects the pharmacokinetics and pharmacodynamics of these agents[18,19]. Binding to SA alters the plasma half-life of a drug, significantly lowers the rate of clearance and changes the volume and the pattern of drug distribution[20]. In addition, SA has a complex binding capacity with small molecules, which makes it difficult to study. There are at least seven hydrophobic binding pockets on the surface of human serum albumin (HSA) that act as universal receptors for drug molecules and modulate cell delivery[21,22]. Due to the high ligand binding capacity of SA, it has become an interest to pharmaceutical companies for drug development although yet to be investigated in respect to small molecule NGF-inhibitor binding. Herein, we report the change in binding affinity for NGF during small molecule interactions with bovine serum albumin (BSA). In this study, BSA was used as a model protein because of its structural homology with HAS[23], and to enable comparison to receptor binding assays used for NGF studies. In the presence of BSA, small molecule-based inhibitors showed an overall increase in binding affinity for immobilized NGF. This increase in binding affinity accounts in part for the discrepancy observed when comparing binding data of small molecule-based inhibitors using cell-free systems, such as SPR, and traditionalin vitromethods. In addition, these findings may provide valuable information necessary for drug delivery and drug design of NGF-inhibitor pain therapeutics. 2 Materials and methods 2.1 Surface plasmon resonance spectroscopy A BIAcore T200 spectrometer was obtained from GE Healthcare Life Science (Piscataway, NJ, USA). Full length (1-118) mouse NGF purified by HPLC from 2.5S NGF (purity >95%) was obtained from Cedarlane Laboratories (Toronto, ON) and coupled to a CM5 sensor chip using standard amine coupling methods. Data was collected at a temperature of 25^oC. Individual compound samples were tested from lowest to highest concentrations, separated by a 15second stabilization period after each sample in each compound series. During each sample cycle, analyte was injected for 60s at a flow rate of 30muL/min. A dissociation period was monitored for 30s after analyte injection before regeneration occurred with a 1.0M NaCl for 120s at a flow rate of 30muL/min to wash any remaining analyte from the sensor chip before running the next sample. Synthetic organic compounds were synthesized by Sussex Research (Ottawa, ON, Canada) and diluted to concentrations ranging from 0.78muM to 200muM using HBS-EP buffer with 0.5% DMSO (0.01M HEPES, 0.15M NaCl, 3mM EDTA and 0.05% v/v Tween 20; pH7.5). To determine binding affinity, the response obtained from each sample run in a series was plotted against concentration using the BIA evaluation software version 2.0 and was evaluated using a two steady-state site binding model as previously described[24]. Data was also analyzed using a heterogeneous ligand model to determine kinetic rate constants. 2.2 Binding of125I-NGF to PC12 cells The iodination of NGF was performed as previously described[25]. The125I-NGF obtained (typically80-120c.p.m./pg) was purified by size exclusion chromatography on a PD10 column (Pharmacia) preequilibrated with HKR buffer (10mM HEPES [pH7.35] containing 125mM NaCl, 4.8mM KCl. 1.3mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, 1g/L glucose, 1g/L BSA). PC12 cells were grown in RPMI with 10% fetal calf serum. Each data point set up in a single tube containing125I-NGF (at the required concentration), 400,000 cells (for a final concentration of 106/mL) and NGF (at 10nM for nonspecific binding) as required. The tubes were incubated for 2h at 4^oC, and 100muL aliquots (providing triplicate data points for each sample) were transferred to 400muL microcentrifuge tubes containing 200muL of 10% glycerol in HKR. Tubes were centrifuged for 2min at 5000rpm, the tip containing the cell pellet was cut off, and radioactivity bound to the cells was determined. 3 Results A series of small molecule NGF inhibitors were screened using SPR spectroscopy for their binding affinity to immobilized NGF. A two-site binding model was applied using BIAevaluation software (Version 2.0). The response obtained due to binding from each analogue concentration was plotted against concentration to determine binding affinity (KD). To determine the effect of BSA on small molecule binding to immobilized NGF, samples were suspended in HBS-EP buffer with 0.5% DMSO in the presence and absence of 1% w/v BSA.Fig. 1represents the two-site steady-state affinity plot for the binding of BVNP-0197 (500-0.78muM) to immobilized NGF. The binding affinity of BVNP-0197 for NGF in the absence of BSA was measured at 21.0muM. The addition of 1% BSA increased the binding affinity of BVNP-0197 for NGF to 0.428muM.Table 1describes the changes in SPR measured binding affinity for all small molecule compounds to immobilized NGF in the presence and absence of BSA. Kinetic rates were determined using a heterogeneous ligand model in the BIAevaluation software (Version 2.0). The marked increase in binding affinity of BVNP-0197 was measured by a decrease in rate of association as well as an increase in rate of dissociation (Fig. 2). The subtracted sensogram of BVNP-0197 (200muM) was chosen to represent the changes in kinetic rates (Fig. 2) however each analyte injection showed similar changes in their respective phases. All small molecule compounds were subjected to kinetics rate analysis which demonstrated the same trend in results (data not shown). Binding saturation studies with125I-NGFin vitrowere completed to measure inhibitory potential for each compound. A half maximal inhibitory concentration (IC50) was determined for each small molecule to determine the concentration by which125I-NGF binding was inhibited by 50% (Table 1). The relationship between SPR high affinity binding of small molecule compounds to immobilized NGF and the respective IC50measured usingin vitro125I-NGF binding was found to be statistically significant when SPR binding was completed in the presence of BSA (F(1,12)=5.070; p=0.0480; R2=0.3364) (Fig. 3). This relationship was not statistically significant when SPR binding was completed in the absence of BSA (F(1,12)=1.643; p=0.2289; R2=0.1411). 4 Discussion Early phase drug discovery is focused on identifying compounds with optimal affinity and selectivity of drug targets. Methods for determining target receptor affinity is being shifted towards cell-free systems which can accurately measure direct binding of a compound to its target receptor without the influence of other identities in the system[26,27]. SPR is one technology available to measure drug specificity to a target protein using a label-free method with high enough sensitivity to look at small molecule compounds (<500Da) binding to larger target proteins (>26kDa)[28,29]. Although drug interactions are often described as a IC50or KDvalue, emphasis on drug kinetics is also ideal for identifying lead compounds during drug development[27,30,31]. A drug with a shorter association phase translates into higher target receptor specificity and likely reduced side effects if used as a therapeutic. In addition, a longer target receptor dissociation phase is generally ideal for reducing therapeutic doses and extending the drug effects. Alternatively, if side effects related to drug toxicity are of concern, a shorter target receptor dissociation phase can be targeted. Small molecule NGF-inhibitors have been an area of focus for nearly two decades as a potential therapeutic option for pain. NGF plays a large role in pain signalling through the TrkA receptor in conditions where an inflammatory pain responses is persistent, such as in autoimmune diseases or arthritic pain[32-34]. Recently, screening for lead small molecule NGF-inhibitors has seen success with cell-free SPR techniques, with identifying novel small molecule compounds with higher specificity for NGF and increased inhibitory potential in altering NGF-TrkA binding than any previously reported compound[24,35,36]. However, with this transition of measuring binding in cell-free systems, a discrepancy exists when comparing values to traditional cell-based techniques. These discrepancies may be a result of a carrier protein influencingin vitrosmall molecule-NGF binding, which is not present during SPR cell-free assays. SA has been identified as a universal drug carrier to aid drug delivery to targeted tissues[18,37,38]. Cell culture protocols, such as125I-NGF binding experiments, require the addition of SA to media to deliver important nutrients to cells, as well as to bind toxins and free radicals reducing cell damage[39]. SA may also play a critical role during small molecule binding to NGFin vitro. The presence of 1% BSA during cell-free analysis of small molecule compounds binding to immobilized NGF measured an overall increase in affinity when compared to binding in the absence of BSA. Kinetic rate analysis determined that in the presence of 1% BSA, small molecule compounds had a decreased rate of association for immobilized NGF in addition to an increased rate of dissociation (Fig. 2). These changes in kinetic rate account for the increase in measured binding affinity and could possibly translate into an increased specificity for NGF with less possible toxic effects if used as therapeutic agents. SPR analysis of small molecule binding to immobilized NGF in the presence of 1% BSA was found to be statistically correlated to values determined through125I-NGF binding (Fig. 3). This relationship is not statistically significant in the absence of BSA. Thus, it is possible that BSA acts as a carrier protein for the small molecules for increased affinity for NGF. It is hypothesized that BSA binds small molecule compounds, enabling a small axial rotation of the compound's structure, placing the small molecule in a conformation ideal for high affinity binding to available NGF. 5 Conclusions SPR cell-free techniques provide an advantage in drug development by eliminating other potentially influencing proteins traditionally found in cell-based binding assays. By introducing proteins independently, SPR allows for a more clear understanding of the binding events which occurin vitroaffecting drug activity. By identifying carrier proteins which participate in small molecule binding to NGF the development of future pain therapeutics will evolve to account for such protein interactionsin vivo. BSA has shown to alter small molecule binding to immobilized NGF in a cell-free system. This shift in binding affinity for NGF in the presence of BSA eliminates the previously observed variance in binding measurements when compared to cell-based assays. These results suggest that BSA plays a critical role in small molecule binding efficacy to NGFin vitro. In addition, these results will allow tailoring future development strategies to account for the influence of carrier proteins to identify therapeutic lead agents. Conflict of interest The authors don't have any conflict of interest. Funding There are no funding sources associated with this research program. References [1] L. Aloe M.L. Rocco P. Bianchi L. Manni Nerve growth factor: from the early discoveries to the potential clinical use J. Transl. 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