Drug-Protein Interactions for Clinical Research by Nucleic Acid Programmable Protein Arrays-Quartz Crystal Microbalance with Dissipation Factor Monitoring Nanoconductometric Assay

Nanoworld Institute, Fondazione EL.B.A. Nicolini (FEN), Largo Redaelli 7, 24020, Pradalunga, Bergamo, Italy Laboratories of Biophysics and Nanobiotechnology (LBN), Department of Experimental Medicine (DIMES), University of Genoa, Via Pastore 3, 16132, Genova, Italy Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University (ASU), Tempe, Arizona 85287, United States


Introduction
Gene-drug (Gottlieb and Altman, 2014;Penrod and Moore, 2014) and protein-drug (Jain, 2004;Witzmann and Grant, 2003) interactions play a major role in the field of molecular pharmacology, as a detailed understanding of these interactions is essential for a proper drug development and delivery. In particular, pharmacodynamics and pharmacokinetics of antiblastics are of high clinical interest (Xie et al., 2014), as cancer is one of the major issues to be still addressed in the field of clinical biomedicine (Robert et al., 2014).
Despite extensive investigation, the etiopathogenesis of GBM is not clear. Some authors speculate that some infectious agents, such as cytomegalovirus (Cobbs, 2013;Thon et al., 2013) or Human Papillomavirus (HPV) (Vidone et al., 2014), may drive the neoplastic process, while other scholars think that occupational exposures to ionizing radiation, the more widespread usage of cellular phones, or a decrease in risk by history of allergies could lead to tumorigenesis. . Others think of incorrect nutritional and eating behaviors (Sandrone et al., 2014). What is known is that only a small percentage of these tumors (less than 1%) is due to Mendelian pathologies, such as neurofibromatosis type 1 and type 2, tuberous sclerosis, Turcot's syndrome, Gorlin syndrome, melanoma-astrocytoma syndrome and Li-Fraumeni syndrome (Malmer et al., 2007;Ostrom et al., 2014). Summarizing, under the same umbrella of GBM many heterogeneous diseases are included, characterized by the involvement of different genetic pathways and by different clinical prognostic outcomes (Patel et al., 2014;Rodriguez-Hernandez et al., 2014;Thakkar et al., 2014).
The current standard of care and treatment for patients with GBM include maximal safe surgical resection, followed by concurrent fractionated Radiation Therapy (RT) to the resection cavity (60 Gy, over 6 weeks) and chemotherapy (with temozolomide (TMZ), followed by adjuvant TMZ) (Stupp et al., 2005;Weathers and Gilbert, 2014).
Despite this highly integrated multimodal, multidisciplinary approach, more than half of the tumors are resistant to this therapy and there is an urgent need to develop novel, effective treatments. Experimental therapies are based on oncolytic herpes simplex virus (Ning and Wakimoto, 2014), administration of monoclonal antibodies (such as bevacizumab, cetuximab, imatinib, gefitinib, erlotinib, cedarinib, sunitinib and vatalanib, among the others), new drugs such as cilengitide, Laser Interstitial Thermal Therapy (LITT), or immunotherapy. For a complete review concerning the different therapeutic options, the reader is referred to (Furnari et al., 2007). Temodar, Temodal and Temcad) is an oral antiblastic, chemically being the imidazotetrazine derivative of the alkylating/methylating agent dacarbazine and it undergoes rapid chemical conversion in the systemic circulation at physiological pH to the active compound, 3-Methyl-(triazen-1-yl) Imidazole-4-Carboxamide (MTIC). TMZ is useful for treating brain tumors, such as the GBM, the relapsed Grade III anaplastic astrocytoma, especially if nitrosourea-and procarbazine-refractory, as well as skin cancers, like the melanoma and the fungoides mycosis/Sézary syndrome (Querfeld et al., 2011). It is currently in evaluation for the treatment of other tumors, such as the relapsed primary CNS lymphoma, recurrent glioma and oligodendroglioma (in the last case, replacing the classical regimen Procarbazine-Lomustine-Vincristine (PCV)). When it binds to the DNA, usually at the N-7 or O-6 positions of guanine residues, it produces O(6)-Methylguanine (O6MG) and this abduct causes the activation of futile DNA Mismatch Repair (MMR), as well as DNA Double-Strand Breaks (DSBs), G(2) arrest and ultimately cell death. The activation of the molecular mechanisms of MMR is quite a complex biological process that required different protein-protein interactions, such as the Mre11/Rad50/Nbs1 (MRN complex), the Proliferating Cellular Nuclear Antigen (PCNA) complex and the gamma-H2AX and 53BP1 foci (Mirzoeva et al., 2006).

TMZ (brand name
Unfortunately, some cells can escape from this mechanisms, producing a protein known as O6alkylguanine DNA Alkyltransferase (AGT) and encoded by the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene. Recently, scientists have been able to find and characterize some biomarkers of resistance to TMZ, such as MLH1, which is also an important marker of survival rate in patients with glioblastoma (Mirzoeva et al., 2006;Querfeld et al., 2011;Shinsato et al., 2013;Stark et al., 2010;von Bueren et al., 2012).
In the last years, the evolution of the nanobiotechnologies applied to proteins, namely proteomics, both structural and functional and specifically the development of more sophisticated protein arrays, has enabled scientists to investigate protein interactions and functions with an unforeseeable precision and wealth of details (Nicolini et al., 2012a;2012b;Nicolini et al., 2013). Moreover, protein arrays can be coupled with label-free approaches: The so-called cell-free protein arrays (Bragazzi et al., 2014b;Dixon, 2008;Fee, 2013;Hunter, 2009;Spera et al., 2013).
In this manuscript, we report and discuss some preliminary results of protein expression of genes related to cancer and in particular to brain tumors and GBM. Experiments have been carried out coupling Nucleic Acid Programmable Protein Array (NAPPA) with a recently improved nanogravimetric apparatus which exploits the Quartz Crystal Microbalance with Frequency (QCM_F) and quartz Crystal Microbalance with Dissipation Monitoring (QCM_D) technologies (Nicolini et al., 2012a;Spera et al., 2013). The selected proteins are BRIP1 and MLH1 and their role and biological roles will be discussed further in this manuscript.
We chose NAPPA since this innovative technology avoids any time-consuming task in the difficult process of obtaining highly purified proteins, relying instead on the production of proteins from high quality supercoiled DNA. For this purpose, complementary DNAs (cDNAs) of selected genes tagged with a C-terminal Glutathione S-Transferase (GST) are spotted on the microarray surface and expressed using a cell-free transcription/translation system (IVTT, in vitro transcription and translation). The newly expressed protein is captured on the array by an anti-GST antibody that have been co-immobilized with the expression clone on the microarray surface.
The advantages and benefits of NAPPA technologies can be briefly summarized : • The demanding process of obtaining highly purified proteins is replaced by a single quick step; furthermore, cDNAs and clones are more easily available • Proteins expressed on the NAPPA arrays are stable, properly folded and biologically, functionally active NAPPA microarrays can be useful in biomarkers discovery and for other clinical applications, such as biosensor development, especially in the effort of moving towards Personalized Medicine. For this task we coupled NAPPA with a new generation of conductometric devices, namely QCM. QCM_D indeed appears a promising tool to study protein-protein interactions especially in the field of oncology, both cellular and molecular (Cheng et al., 2012).
To the best of our knowledge, we coupled for the first time QCM_D with NAPPA technology for biomedical applications in the field of neuro-oncology. Moreover, there are few biosensors developed for GBM, usually for cellular sensing (Beljebbar et al., 2010;Brasuel et al., 2001;Chen et al., 2008;Desai et al., 2006;Manning et al., 1998;Trevin et al., 1998;Valero et al., 2010;Zakir Hossain et al., 2007). The objective of the present research regards the analysis of protein-drug and multiple protein-drug interaction towards potentially useful clinical applications, namely in the field of cancer studies.
Clinical implications are also envisaged and addressed.

QCM_D Conductometer
Nanogravimetry makes use of functionalized piezoelectric Quartz Crystals (QC), which vary their resonance frequency (f) when a mass (m) is adsorbed to or desorbed from their surface. This is well described by the well-known Sauerbrey's equation: where, f 0 is the fundamental frequency, A is the surface area covered by the adsorbed molecule and ρ and l are the quartz density and thickness, respectively. Quartz resonators response strictly depends on the biophysical properties of the analyte, such as the viscoelastic coefficient. The dissipation factor (D) of the crystal's oscillation is correlated with the softness of the studied material and its measurement can be computed by taking into account the bandwidth of the conductance curve 2Γ, according to the following equation: where, f is the peak frequency value.
In our analysis we introduced also a "normalized D factor", D N , that we defined as the ratio between the halfwidth half-maximum (Γ) and the half value of the maximum value of the conductance (G max ) of the measured conductance curves : D N is more strictly related to the curve shape, reflecting the conductance variation (Bragazzi et al., 2014a;Spera et al., 2013).

NAPPA Experiments
The QCM_D instrument was developed by Elbatech (Elbatech srl, Marciana-LI, Italy). The quartz was connected to an RF gain-phase detector (Analog Devices, Inc., Norwood, MA, USA) and was driven by a precision DDS (Analog Devices, Inc., Norwood, MA, USA) around its resonance frequency, thus acquiring a conductance versus frequency curve ("conductance curve") which shows a typical Gaussian behaviour. The conductance curve peak was at the actual resonance frequency while the shape of the curve indicated how the viscoelastic effects of the surrounding layers affected the oscillation. The QCM_D software, QCMAgic-Q5.3.256 (Elbatech srl, Marciana-LI, Italy) allows to acquire the conductance curve or the frequency and dissipation factor variation versus time. In order to have a stable control of the temperature, the experiments were conducted in a temperature chamber. Microarrays were produced on standard nanogravimetry quartz used as highly sensitive transducers. The QC expressing proteins consisted of 9.5 MHz, AT-cut quartz crystal of 14 mm blank diameter and 7.5 mm electrode diameter, produced by ICM (Oklahoma City, USA). The electrode material was 100 Å Cr and 1000 Å Au and the quartz was (Nicolini et al., 2012b;Spera et al., 2013). The NAPPA-QC arrays were printed with 100 spots per QC.
Quartzes gold surfaces were coated with cysteamine to allow the immobilization of the NAPPA printing mix. Briefly, quartzes were washed three times with ethanol, dried with Argon and incubated over night at 4°C with 2 mM cysteamine. Quartzes were then washed three times with ethanol to remove any unbound cysteamine and dried with Argon. Plasmids DNA coding for GST tagged proteins were transformed into E. coli and DNA were purified using the NucleoPrepII anion exchange resin (Macherey Nagel). NAPPA printing mix was prepared with 1.4 µg uL −1 DNA, 3.75 µg uL −1 BSA (Sigma-Aldrich), 5 mM BS3 (Pierce, Rockford, IL, USA) and 66.5 µg polyclonal capture GST antibody (GE Healthcares). Negative controls, named master mix (hereinafter abbreviated as "MM"), were obtained replacing DNA for water in the printing mix. Samples were incubated at room temperature for 1 h with agitation and then printed on the cysteamine-coated gold quartz using the Qarray II from Genetix. In order to enhance the sensitivity, each quartz was printed with 100 identical features of 300 microns diameter each, spaced by 350 microns center-to-center. The human cDNAs immobilized on the NAPPA-QC were: MLH1 (mutL homolog 1) and BRIP1 (BRCA1 interacting protein C-terminal helicase 1).
Gene expression was performed immediately before the assay, following the protocol described in . Briefly, IVTT was performed using HeLa lysate mix (1-Step Human Coupled IVTT Kit, Thermo Fisher Scientific Inc.), prepared according to the manufacturers' instructions.
The quartz, connected to the nanogravimeter inside the incubator, was incubated for 10 min at 30°C with 40 µL of HeLa lysate mix for proteins synthesis and then, the temperature was decreased to 15°C for a period of 5 min to facilitate the proteins binding on the capture antibody (anti-GST). After the protein expression and capture, the quartz was removed from the instrument and washed at room temperature, in 500 mM NaCl PBS for 3 times. The protocol described above was followed identically for both negative control QC (the one with only MM, i.e., all the NAPPA chemistry except the cDNA) and protein displaying QC.
After protein expression, capture and washing the QCs were used for the interaction studies QC displaying the expressed protein was spotted with 40 µ l of drug solutions in PBS at increasing concentrations at 22°C.
Reproducibility of the experiments was assessed computing the Coefficient of Variation (CV, or σ*), using the following equation: where, σ is the standard deviation and µ is the mean. We also tested the possibility to analyze drug-protein interactions in QC displaying multiple proteins. For this aim, we co-printed cDNA for BRIP1&MLH1 on a single QC. We analyzed the interaction response to TMZ on both NAPPA-expressed QCs.
We analyzed the interaction between BRIP1, MLH1 and TMZ drug solutions at different concentrations to analyze the binding kinetics after protein expression and capture the expressing QC was spotted, in sequence, with 40 µL of increasing Temozolomide solutions of concentration: 1, 2, 5, 10, 20, 50, 100 and 200 µg mL −1 . As negative control we analyzed the interaction between BRIP1/FANCJ, a helicase initially linked to breast cancer (Cantor and Xie, 2010) and to Fanconi anemia and TMZ, while MLH1, which is a protein involved in DNA mismatches repair, is known to interact with TMZ. We analyzed the conductance curves acquired in NAPPA-QCs in different steps of the expressing and capturing process: After the addition of human IVTT lysate at 30°C ("IVTT addition"), i.e., prior protein expression; after 10 min from the addition of human IVTT lysate, i.e., after protein expression ("IVTT addition 10 min"); after the final washing process with PBS ("Post-wash").

Results and Discussion
In Fig. 1 are reported the conductance curves of increasing concentrations of TMZ spotted on quartz blanks, while in Fig. 2 are shown the conductance curves of quartz carrying MLH1 gene being expressed and thereafter interacting with TMZ solutions at increasing concentrations are reported. Figure 3 shows the response to increasing concentrations of TMZ: Since MLH1 interacts with the drug, this response is linear up to 200 µg mL −1 . Figure 4 shows the conductance curves for NAPPA-QCs expressing BRIP1. We analyzed the interaction among BRIP1 and TMZ, verifying that the protein does not interact with the drug. Figure 5 reports the conductance curves for NAPPA-QCs carrying BRIP1&MLH1 being co-immobilized. Figure 6 shows the response to increasing concentrations of TMZ: We reproduce the behavior shown in Fig 4, even though with an exponential fit.
These data pointed to a unique conductance curve shape for each protein and suggested the possibility to identify the expressed proteins by QCM-D even when combined on the same expressing QC (Fig 7).
In Table 1-3 are reported the main parameters of the conductance curves of Fig 2, 4 and 5, respectively.
In Table 4 and 5 are reported the two multiple regression models that have been used to predict the behavior of the multi-gene experiment (MM_BRIP1&MLH1 interacting with TMZ). Conductance curves were collected in different steps of NAPPA protocol. b f is peak frequency, Γ is the half-width half-maximum (HWHM) and G max is the maximum conductance. c D factor and D N (computed as D N = 2Γ/G max ) normalized D factor Conductance curves were collected in different steps of NAPPA protocol. b f is peak frequency, Γ is the half-width half-maximum (HWHM) and G max is the maximum conductance. c D factor and D N (computed as D N = 2Γ/G max ) normalized D factor Conductance curves were collected in different steps of NAPPA protocol. b f is peak frequency, Γ is the half-width half-maximum (HWHM) and G max is the maximum conductance. c D factor and D N (computed as D N = 2Γ/G max ) normalized D factor

Conclusion
In this study, we introduced a new application of our previously described NAPPA-based nanoconductometric sensor, (Nicolini et al., 2012a;2012b;Nicolini et al., 2013), which combined with Mass Spectrometry using SNAP arrays (Nicolini et al., 2013) has been extended to cancer studies (Bragazzi et al., 2014a), and in this context has been used to clinically screen patients respondent to TMZ from those refractory to this drug. We performed a positive control (TMZ plus MLH1 protein), a negative control (TMZ plus BRIP1 protein) and a multi-gene experiment (TMZ plus BRIP1&MLH1 being co-expressed), showing that we are able to properly perform pharmacoproteomics tasks, discriminating each protein and drug unique conductance curve as well as their interactions, even in the presence of multi-proteins being immobilized. Moreover, in the last part of our paper, we used a multiple regression model in order to predict the behavior of TMZ when exposed to BRIP1&MLH1 co-expressed and we showed that we are able to predict the drug-protein interaction profile with a good regression coefficient.

Funding Information
This project was supported by grants to Professor Claudio Nicolini of the University of Genova, Italy by the FIRB Nanobiosensors (ITALNANONET RBPR05JH2P_003) and by a grant Funzionamento to Fondazione ELBA Nicolini from MIUR (Italian Ministry for Research and University).

Author's Contributions
CN and EP designed and carried out the original experiments, RS and NLB performed the QCM_D measurements, NLB and CN analyzed the data and wrote the paper finalized by CN alone.

Ethics
All the experiments are in vitro and therefore do not require ethical approval.