Cite this article: Sharma S, Yadav VK, Yadav A (2018) Non-Covalent Carriage in the Fab Region of the Monoclonal Antibody Trastuzumab May Improve Small Molecule Anticancer Chemotherapy. J Drug Des Res 5(2): 1068.
*Corresponding authorArpita Yadav, Department of Chemistry, University Institute of Engineering and Technology, Chhatrapati Shahu Ji Maharaj University, Kanpur 208024, India, Tel: (91)9532692209; Email:
Non-Covalent Carriage in the Fab Region of the Monoclonal Antibody Trastuzumab May Improve Small Molecule Anticancer ChemotherapySweta Sharma1, Veejendra Kumar Yadav2 and Arpita Yadav1*1Department of Chemistry, Chhatrapati Shahu Ji Maharaj University, India2Department of Chemistry, Indian Institute of Technology, India
Abstract
Recent advances in medicine suggest a combination therapy of immunotherapeutic and chemotherapeutic agents to be clinically successful in treating cancer patients, especially the cases of breast cancer where HER2 receptor is over expressed in about 30% of breast cancers. Recent experimental results indicate that immunotherapy enhances the efficiency of chemotherapeutic drug, perhaps by acting as its carrier. Recent success in crystallizing cyclic peptides inside humanized antibodies led us to demonstrate a plausible mechanism at the molecular level in our previous work. The generalized effort on a database of anticancer agents is described here. While authenticating the generality of the concept, special consideration has been given to drugs with severe side effects. A subset of compounds with severe side effects was chosen from a prepared database of clinically used anticancer drugs. The study depicts internalization of all compounds in antibody utilizing standard docking protocol. Further filtering was done based on more accurate Quantum polarized ligand docking (QPLD) to reconfirm previously identified descriptors for non-covalent carriage of anticancer agents and their delivery at target. Molecular dynamics simulations have been performed on the four best internalized compounds to show smooth carriage and negligible probability of premature expulsion of cargo. Affirmation of identified descriptors for carriage, points out the suitability of these chemotherapeutic agents for combination therapy enhancing their efficacy and reducing side effects. However, these results remain to be verified by in vitro or clinical studies.
INTRODUCTIONAn ever increasing number of cancer patients has accelerated
global attempts to find a successful cure for this fatal disease. One of such recent attempts is immunotherapy which uses our own immune system to fight against cancer [1]. Although this was not as successful as initially predicted, it did seem to enhance the efficacy of small chemotherapeutic agents on co-administration [2,3]. This brought about new hopes for combination therapy to reduce the severely irritating cytotoxic effects of chemotherapy. In our recent work [4], we have predicted non-covalent carriage of anticancer agents by antibody trastuzumab. Along with the antibody the anticancer agent is also targeted towards HER2 receptor on dividing cells and therefore enhanced efficacy may be expected. We also identified certain descriptors for screening the compounds for efficient carriage.
This is a follow up study to show the general applicability of such a combination treatment. In this work, we have screened a database of anticancer agents for their possible non-covalent carriage through humanized antibody trastuzumab, with the aim of illustrating and predicting their enhanced efficacy, when co-administered with an immunotherapeutic agent. We believe that
this study will help in enhancing the efficacy of small anticancer agents and, at the same time, will also reduce their cytotoxicity due to targeted delivery, through an immunotherapeutic agent. It is to be further emphasized that non-covalent carriage is a non-destructive technique with improved bioavailability.
METHODOLOGYFirst of all, a database of anticancer compounds administered
intravenously and showing severe side effects and cytotoxicity issues (Table 1) [5-29] was prepared.
These anticancer agents were prepared for docking studies. The Ligprep module of Schrodinger software [30] was used for preparing the anticancer chemotherapeutic agents. Epik [31] was used to predict the ionized state of the compound at physiological pH. The compounds were then docked in the middle space of humanized antibody trastuzumab (pdb Id: 4IOI) [32] utilizing standard precision docking module of Glide. The flexible ligand docking procedure was the same as described in our previous study [4]. In this work, a multistep filtering protocol was followed to identify compounds with the most appropriate features for carriage through antibody starting with a database of
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anticancer compounds. This work also demonstrates the general applicability of combination therapy (immunotherapeutic agent with chemotherapeutic agent) to enhance the efficacy of otherwise severely cytotoxic chemotherapeutic agent. The overall procedure is shown in Figure 1. The utility of combination therapy lies in enhancing the efficacy and reducing the side effects of clinically used chemotherapeutic agents. The multistep filtering protocol is briefly given in the following sections:
Standard precision docking
The X-ray structure of humanized antibody trastuzumab [32] has recently been released where a small cyclic peptide is crystallized within its middle space. This structure was taken and hydrogens were added. The protein was prepared for docking studies. Care was taken to ensure that all ionizable residues were in a proper form, as in physiological pH. Low energy conformations of all the compounds in the database were generated using ligprep and Macromodel modules [33] of Schrodinger software with Maestro interface. Ten lowest energy conformations were filtered for docking. The cyclic peptide was then removed from the antibody. The target grid was placed selecting 15-20 residues from the heavy chain and 6-7 residues from light chain immediately surrounding the middle space in the Fab region of the antibody. The middle space in the Fab region of antibody has been chosen for non-covalent carriage of anticancer agents as it does not hinder with the antigen-binding site which must remain unobstructed to drive the antibody towards HER2 receptor. Also,
the middle space facilitates smuggling the chemotherapeutic agent to target without much probability of premature expulsion. The grid was then validated by redocking the cyclic peptide in the middle space. It was ensured that antigen binding site remained unaltered and unoccupied. Fifty poses were generated with each filtered conformation. Glide flexible ligand, rigid receptor docking with post docking minimization [34-36] was then performed on lowest energy conformations. The terms in the glide docking score function are defined as follows:
GScore = 0.05 vdW + 0.15 Coul + Lipo + H Bond + Metal + Rewards + Rot B + Site
Where vdW = vander Waals energy, Coul = Coulombic energy, Lipo = lipophilic term derived from hydrophobic grid potential, H bond = the hydrogen bonding term, metal = metal binding term, rewards are the rewards and penalties that cover other terms than those explicitly mentioned like buried polar groups, hydrophobic enclosure, amide twists, etc., rot B is the penalty for freezing rotatable bonds, and site is the polar but non hydrogen bonding interactions in the active site [34]. The best eight docking score compounds were segregated for next round of Quantum Polarized Ligand Docking (QPLD) and MM/GBSA calculations after solvation.
Quantum polarized ligand docking
QPLD combines Glide docking with the quantum mechanical ab initio accuracy of QSite module [37]. QSite performs single
Table 1: Small molecule chemotherapeutic agents with severe side effects.
S. No. Anticancer drugs Chemical formula Mode of administration
point energy calculations on each complex pose, previously generated by single precision Glide docking. It treats the ligand with ab initio method and derives partial atomic charges using electrostatic potential fitting. The ligand is then re-docked utilizing charges calculated by QSite. QPLD algorithm then returns the energetically favourable poses. Best ten poses based on re-scoring for each of the eight compounds were selected. Selected poses were subjected to solvation using implicit solvent. Interaction energies for the poses were then calculated utilizing combined Molecular mechanics-generalized Born surface area (MM-GBSA) approach.
MM/GBSA binding energy analysis
MM/GBSA binding energy calculations have been performed utilizing the Prime module of Schrodinger software [38]. This approach utilizes the molecular mechanics OPLS 2005 force field coupled with generalized Born surface area continuum solvent model for the prediction of solvation energies of complex and the two fragments [39]. The binding free energy (∆Gbind) of each ligand is then calculated as:
ΔGbind = ΔEMM + ΔGsol + ΔGSA
ΔEMM is the difference in minimized energies of complex and
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Table 3: MM/GBSA binding energies for selected chemotherapeutic agents.
MM/GBSA Binding Energy and its different contributions Energies are given in kcal/mol
ΔGsol is the difference in solvation energies of complex and sum of solvation energies of antibody and cargo drug.
ΔGSA is the difference in surface area energies of complex and sum of surface area energies of antibody and cargo drug.
The ligand strain energy correction was also considered. Further filtering was done based on descriptors identified in our previous study for optimum non-covalent carriage.
Filtering based on desired descriptors for non-covalent carriage
After understanding the non-covalent carriage feasibility of small molecule anticancer agents from the database; the last round of filtration was carried out to identify the most suitable compounds for combination therapy and to re-assert descriptors identified in our previous study for non-covalent carriage. Out of eight compounds subjected to QPLD and MM/GBSA calculations, only four compounds best fulfilling the desired descriptors were chosen after careful analysis of their electrostatic and hydrophobic interactions with Fab region of antibody. Small buried hydrophobic surface area of complex has also been considered as an indication of good internalization of the drug. A grid of points equidistant from each atom of the molecule was taken and the number of solvent accessible points were determined. All points were checked against the surface of neighbouring atoms to determine whether they were buried. The buried hydrophobic surface area of complex was then taken as a parameter in the filtration process.
Molecular dynamics (MD) simulation studies
The best binding energy complex has been chosen for each of the four drugs and subjected to MD simulations to judge the stability, dynamical behavior of complex with time and any chances of premature expulsion of drug. All atom solvent was incorporated in a grid of 10×10×10 Å around the antibody using the SPC model. Orthorhombic boundary conditions were applied. The minimized complex was subjected to simulated annealing followed by dynamics at 300 K for 25 ns as per standard MD protocol. Shaw’s Desmond software [40] has been used to perform dynamics. NPT (isothermal-isobaric) ensemble was chosen utilizing the Martyna-Tobias-Klein barostat method [41]. The reversible reference system propagator algorithms (RESPA) integrator was used with 2 fs time step for near and bonded atoms, and 6 fs time step for far atoms as per default settings [42]. Coulombic interaction cut off was set at 9.0 Å. To understand the fulfilment of key binding descriptors identified in our earlier work, the hydrogen bonds and other close contacts formed throughout the simulation were monitored and analyzed. The Ligand-Protein interaction maps (L-P maps) generated gave the strength of the interactions. The importance of the descriptor is highlighted through these interaction maps. The RMSD plots indicate the quality of dynamics and the stability of the complex. The MD simulations settings are largely similar to our previous study [4], but the docking procedure was carried out at the more accurate level, utilizing quantum mechanically treated ligand, with accurate atomic charges for proper polarization and protein induced fit.
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RESULTS AND DISCUSSIONAs mentioned in the procedure described above; standard
precision docking was performed on all compounds in the collected database (Table 1) of anticancer agents. Twenty compounds were dockable in the middle space of Fab region of antibody. The choice of middle space carriage lets the monoclonal antibody be targeted to HER2 receptor simultaneously facilitating delivery of chemotherapeutic agent also to rapidly dividing cells. Docking results for these compounds are summarized in Table 2. The results indicate that most of the chemotherapeutic agents are dockable in the Fab region of antibody trastuzumab. Results of best two poses obtained are shown in Table 2. Best eight dockable compounds were subjected to rescoring by QPLD for more accurate docking results. Bleomycin was not rescored as the pose indicates improper docking and partial internalization of drug due to its larger size. Results after rescoring are also given in Table 2. Figures 2a and 2b display the best docked poses for these drugs, along with the buried hydrophobic surface area of the complex. Rescoring eliminates any false positives, although the grid was validated by re-docking the crystallized cyclic peptide, and the middle space of Fab region is well defined and away from the antigen binding site. Figure 2 indicates that the drugs can be nicely accommodated in the middle space of the Fab region, with buried hydrophobic surface area of complex less than 80.0 Å2. Low buried hydrophobic surface area is a measure of good internalization of drug. The buried hydrophobic surface area for Bleomycin pose was observed to be much higher (224.78 Å2), when compared to other cases (Figure 2) indicating partial internalization, poor carriage and chances of premature expulsion of drug.
The best ten poses of each of the eight compounds obtained after QPLD were subjected to MM/GBSA binding energy evaluations after introduction of implicit solvent around the complex. MM/GBSA binding energies along with their different contributions are shown in Table 3. Two poses for each drug, corresponding to best binding energies, were tabulated. Maximum binding energy contribution comes from electrostatic interaction followed by van der Waals interaction contribution and lipophilic energy. For further filtering of the best suited chemotherapeutic agents, for combination therapy with immunotherapeutic agents, we considered the descriptors identified by us in our earlier work for good non-covalent carriage inside Fab region of antibody trastuzumab. Reiterating the descriptors identified, an indole-indoline type system containing compound may be non-covalently carried by an antibody and delivered at target. A positively charged drug in proper conformation, so as to form efficient salt bridges with neighboring negatively charged residues is desirable for efficient carriage through antibody. The drug must also interact favorably with neighboring hydrophobic residues of heavy chain. After internalization of drug, the antibody-drug complex must show a small buried hydrophobic surface area indicative of good internalization. Figure 4 shows ligand-interaction maps for the eight filtered compounds, which indicate the extent of polar and hydrophobic interactions with the Fab region of antibody. Daunomycin and dauxorubicin (Figure 3a) both show electrostatic interactions with glu155 of heavy chain. Daunomycin also shows salt bridge with glu165 of light chain. Mitoxantrane and epirubicin also show similar electrostatic
interactions with glu155 and glu165 (Figure 2b). Other than these, all the four drugs show hydrophobic interactions with neighboring residues of heavy chain of Fab region of antibody, which further helps in their non-covalent anchorage within the middle space of Fab region.
The rest of the chemotherapeutic agents, the interactions of which are shown in Figures 3c,d, show lesser interactions with the Fab region. Plicamycin shows a significant number of interactions and a similar buried hydrophobic area, but does not show strong electrostatic interactions with glu155 or glu165 as it is a neutral drug. Considering the fulfillment of the above descriptors, four chemotherapeutic agents, namely daunomycin, dauxorubicin, mitoxantrane and epirubicin, were selected for MD analysis study to judge the extent to which the non-covalent contacts are maintained for extended periods of time to avoid any premature expulsion of drug. Twenty five ns trajectories were evaluated as per the procedure described in the methodology section. Figure 3 shows contact maps for selected drugs highlighting the important polar and non polar (hydrophobic) contacts, salt bridges that remain intact for most of the simulation time and are responsible for the non-covalent carriage of drug by the antibody. It can be seen clearly from Figure 4 that polar interactions are predominant and play an important role in the non-covalent carriage and targeted delivery of chemotherapeutic agent with the immunotherapeutic agent. The polar interactions in general are maintained for longer durations as compared to hydrophobic interactions. Further support for good carriage comes from low root mean square deviations (RMSD) of atomic positions during simulation (Figure 5) which indicates that the chemotherapeutic agent remains internalized and is not prematurely expelled. Figure 5 also compares the RMSD of drugs found to be most suited for non-covalent carriage in this study with the RMSD for vincristine, which was found to be ideal for non-covalent carriage in our previous study. Comparison of RMSD also suggests that daunomycin, dauxorubicin, mitoxantrane and epirubicin are most suited for targeted delivery. With the help of antibody, the chemotherapeutic agent is also targeted to tumor cells. Such non-
Figure 1 A multi-step screening protocol demonstrating the generalized suitability of small molecule anticancer drugs for combination therapy with immunotherapeutic agents.
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Figure 2a Molecular surface of antibody-chemotherapeutic drug complex.
Figure 2b Molecular surface of antibody-chemotherapeutic drug complex.
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Figure 3a Ligand interaction maps showing polar and hydrophobic interactions with neighbouring Fabregion residues of antibody Trastuzumab. Figure 3a: Ligand interaction maps for Daunomycin and Dauxorubicin.
Figure 3b Ligand interaction maps for Mitoxantrane and Epirubicin.
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Figure 3c Ligand interaction maps for Plicamycin and Teniposide.
Figure 3d Ligand interaction maps for Vindesine and Vinblastine.
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Figure 4 Chemotherapeutic drug-antibody Fab region contact map for MD simulation period (The percentage indicated on the contact is the strength of interaction.)
Figure 5 Root mean square deviation (RMSD) in the position of heavy atoms during MD simulation of internalized chemotherapeutic agents ideal for combination therapy with immunotherapeutic agents.
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covalent delivery enhances bioavailability of the anticancer agent and reduces its adverse side effects.
CONCLUSIONIn this study, non-covalent carriage and targeted delivery of
anticancer agents through antibody trastuzumab was studied utilizing molecular modeling and docking studies. Some of the previously identified descriptors for smooth carriage were re-asserted. The study emphasizes the synergistic effects of combination therapy of an immunotherapeutic agent with a chemotherapeutic agent in improving the efficacy of small molecule anticancer agents through their non-covalent carriage. This study is expected to aid in improving the bioavailability, and reducing the toxic effects of chemotherapeutic agents. The study also corroborates similar experimental findings and provides a molecular level insight for the enhanced efficacy of chemotherapeutic agent on co-administration with an antibody. However, further biological studies and clinical trials are required to justify and promote a new avenue treatment strategy comprising of co-administering chemotherapeutic agent and monoclonal antibody trastuzumab.
support (Project no. EMR/2016/000769) from Science and Engineering Research Board (SERB), Department of Science and Technology, New Delhi, and infrastructural support from CSJM University, Kanpur. Ms. Sweta Sharma is thankful to SERB for Research Fellowship. Prof. Veejendra Kumar Yadav gratefully acknowledges financial support from Council of Scientific and Industrial Research (CSIR), New Delhi.
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Sharma S, Yadav VK, Yadav A (2018) Non-Covalent Carriage in the Fab Region of the Monoclonal Antibody Trastuzumab May Improve Small Molecule Anticancer Chemotherapy. J Drug Des Res 5(2): 1068.
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