Structural Approach To Identify a Lead Scaffold That Targets the

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A Structural Approach to Identify a Lead Scaffold that Targets the Translesion Synthesis Polymerase Rev1 Radha Charan Dash, Zuleyha Ozen, Alessandro A. Rizzo, Socheata Lim, Dmitry M. Korzhnev, and M. Kyle Hadden J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00535 • Publication Date (Web): 05 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Journal of Chemical Information and Modeling

A Structural Approach to Identify a Lead Scaffold that Targets the Translesion Synthesis Polymerase Rev1

Radha Charan Dash1, Zuleyha Ozen,1 Alessandro A. Rizzo,2 Socheata Lim,2 Dmitry M. Korzhnev,2 M. Kyle Hadden*1

1Department

of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269, USA

2Department

of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT 06030, USA

ACS Paragon Plus Environment

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ABSTRACT Translesion synthesis (TLS) is a mechanism of replication past damaged DNA through which multiple forms of human cancer survive and acquire resistance to first-line genotoxic chemotherapies. As such, TLS is emerging as a promising target for the development of a new class of anti-cancer agents. The C-terminal domain of the DNA polymerase Rev1 (Rev1-CT) mediates assembly of the functional TLS complex through protein-protein interactions (PPIs) with Rev1 interacting regions (RIRs) of several other TLS DNA polymerases. Utilizing structural knowledge of the Rev1-CT/RIR interface, we have identified the phenazopyridine scaffold as an inhibitor of this essential TLS PPI. We demonstrate direct binding of this scaffold to Rev1-CT and the synthesis and evaluation of a small series of analogues have provided important structureactivity relationships for further development of this scaffold. Furthermore, we utilized the umbrella sampling method to predict the free energy of binding to Rev1-CT for each of our analogues. Binding energies calculated through umbrella sampling correlated well with experimentally determined IC50 values, validating this computational tool as a viable approach to predict the biological activity for inhibitors of the Rev1-CT/RIR PPI.


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1. INTRODUCTION Platinating and alkylating agents are standard first-line chemotherapeutic drugs for multiple tumor types; however, the development of acquired resistance and dose-dependent side effects limits their long-term efficacy1-3. Recent studies have demonstrated that translesion synthesis (TLS) is an important cellular mechanism that allows error-prone replicative bypass of DNA adducts formed by these agents, helping cancer cells survive genotoxic chemotherapy and mediating acquired resistance against standard anti-cancer regimens4-5. In humans, multiple TLS DNA polymerases have been identified that belong to A, B, X and Y families6. The Y-family polymerases Rev1, Polη, Polι, Polκ and the B-family polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) are responsible for the replicative bypass of the majority of DNA lesions in the process of Rev1/Polζ-dependent TLS7-8. Rev1 is a Y-family DNA polymerase with limited deoxycytidyltransferase activity9 whose primary function is to serve as a scaffold for assembly of the multi-protein TLS complex and regulate recruitment of other TLS polymerases through multiple protein-protein interactions (PPIs)7-8. Most of these essential PPIs with other TLS DNA polymerases are mediated through the C-terminal domain of Rev1 (Rev1CT). This versatile domain can bind Rev1-interacting regions (RIRs) from the Y-family polymerases Polη, Polι, Polκ and the PolD3 subunit of the B-family polymerase Pol ζ on one face, while simultaneously binding the regulatory subunit Rev7 of Polζ on the opposite face10-15. Multiple recent studies have detailed the role of Rev1 in mediating cancer resistance to genotoxic chemotherapies. Reduced expression of Rev1 resulted in a significant decrease in both DNA mutation rate and the development of resistance to genotoxic agents in cellular models of ovarian and lung cancer, B-cell lymphoma, and fibrosarcoma16-18. In addition, Rev1 knockdown in these cell lines increased their susceptibility to cisplatin treatment. Taken together, these studies



demonstrate the essential nature of Rev1 in mutagenic Rev1/Polζ-dependent TLS and highlight the anti-cancer potential of small molecules that can successfully disrupt TLS at the level of Rev1. We recently utilized a de novo biochemical screening approach to identify the first small molecules that inhibit TLS in human cancer cells through disruption of the Rev1-CT/RIR PPI18. To continue our identification of small molecule scaffolds that inhibit TLS, we utilized a structurebased computational approach to explore the key intermolecular interactions that govern Rev1-CT PPIs and applied our understanding of these interactions to design a novel lead scaffold that binds Rev1-CT and disrupts the Rev1-CT/RIR PPI. In addition, we have integrated computational and experimental approaches to design and validate additional analogues of this scaffold as inhibitors of this TLS PPI.

2. MATERIALS AND METHODS 2.1. Initial PPI analysis and lead compound design. 2.1.1. Molecular dynamics. To explore the binding energetics for each individual amino acid residue involved in the PPI between Rev1-CT and polκ, the three-dimensional co-structure generated from NMR (PDB code 2LSI) was subjected to molecular dynamics (MD). The general AMBER force field (GAFF)19 was loaded to generate the force field parameters and for all ligands, the HF/6-31G* RESP atomic charges20 were calculated with the antechamber module. The Rev1CT protein and polκ peptide were parameterized by AMBER ff99SB force field21. To neutralize the charge of the system, sodium counter ions were placed to grids with the largest negative coulombic potentials around the protein, and the whole system was immersed in the rectangular box of TIP3P water molecules. The water box extended 8 Å away from any solute atoms. In molecular minimization and molecular dynamics simulations, particle mesh Ewald (PME) was


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employed to treat the long-range electrostatic interactions22. Before MD simulations, the complexes were gradually relaxed using 10,000 cycles of minimization procedure [500 cycles of steepest descent23-24 and 9,500 cycles of conjugate gradient minimization25 After minimization, MD simulations in the NPT ensemble with a target temperature of 298 K and a target pressure of 1 atm were performed. The SHAKE procedure26 was employed to constrain all hydrogen atoms and the time step was set to 2.0 fs. Before the actual MD simulations, the system was gradually heated in the NVT ensemble from 10K to 298K over 500 ps. Initial velocities were assigned from a Maxwellian distribution at the starting temperature. MD samplings at 10 ns were performed for the complex. 2.1.2. MM/GBSA calculations and per residue interaction. All MM/GBSA calculations and per residue interaction studies were performed as described previously27. 2.1.3. Molecular docking and E-pharmacophore generation. The Rev1-CT model from PDB code 2LSI was prepared for docking with the protein preparation wizard module from Schrödinger Suite 2015. The grid box was generated around the amino acids interacting with the RIR region of polκ. The Glide XP docking module was used for the docking study and visualization of the docked results. The docking conformer of the two FF residues along with the binding pose was utilized as the input for E-pharmacophore mapping28. E-pharmacophores were constructed by mapping the energetic terms from Glide XP scoring29 function onto the center of the interacting ligand atom. All ligand structures were built using the Maestro module of the Schrödinger Suite package and energy minimized using Macromodel with the OPLS_2005 force field. The remaining parameters were left at their default settings.



2.2. Post-Experimental Computational Studies. 2.2.1. Absolute free binding energy calculation. In order to explore the absolute free binding energy of all the compounds complexed with Rev1-CT, the potential of mean force (PMF) was calculated by umbrella sampling molecular dynamics simulation and the Weighted Histogram Analysis Method (WHAM)30-31. Taking the Glide XP docking conformation of all the compounds as our initial conformation, we performed this computational study in Gromacs 2016 by following four steps: (i) generate a series of configurations along a single degree of freedom (reaction coordinate), (ii) extract frames from the trajectory in step 1 that correspond to the desired center of mass (COM) spacing, (iii) run umbrella sampling simulations on each configuration to restrain it within a window corresponding to the chosen COM distance, and (iv) utilize WHAM to extract the PMF and calculate ΔG. The ligand-protein complex was manually oriented in such a way that it provides the ligand space for pulling simulations to take place along the x-axis. The charge parameters of all compounds were determined by an ab initio molecular orbital program at the HF/6-31G* level. We employed AMBER 99SB force field parameters for proteins and the TIP3P model for water. Next, we immersed each protein–ligand complex in a TIP3P water rectangular box, with a solvation of at least 10.0 Å from the protein surface. The whole system was neutralized by adding the appropriate number of Na+ and Cl- counter ions. The short-range non-bonded interactions were cut off at 1.4 nm, with long-range electrostatics calculated using the particle mesh Ewald (PME) for all simulations. The energy minimizations of the protein–ligand complexes were performed by the steepest descent method (50,000 steps) with an energy step size of 0.01. Next, the equilibration was performed for MD simulations (0.5) ns at 300 K and 1.0 bar. Following equilibration, restraints were removed from the protein-ligand complex and the protein was used as an immobile reference for the pulling simulations. The ligand was pulled away from Rev1-CT


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along the x-axis over 2 ns, using a force constant of 1000 kJ mol−1 nm−2 and a pull rate of 0.003 nm ps−1. Following the pulling simulations, a final COM distance between Rev1-CT and compound of approximately 3 nm was achieved. From these trajectories, snapshots (reaction coordinates) were collected to generate the starting configurations for the umbrella sampling windows. Different snapshots (15-20, depending on the compound) were collected at a window spacing from 0.1 - 1.2 nm (15 snapshots) and 0.1 - 1.5 nm (20 snapshots). In each window, 50 ns of MD was performed for a total simulation time of 1000 ns utilized for umbrella sampling. Sufficient overlap between the sample windows was validated by the histogram from the adjacent windows (Figure S5). Analysis of the results was performed using WHAM. 2.3. Biological protocols 2.3.1. Protein expression and purification. The Rev1 C-terminal domain (Rev1-CT; residues 1158-1251) was expressed in E. coli BL21(DE3) cells and purified following established protocols 11-12.

For NMR binding studies,

15N

labeled and unlabeled Rev1-CT (expressed in M9 and LB

media, respectively) were exchanged into 50 mM NaH2PO4, 100 mM NaCl, 10% D2O, pH 7.0 buffer. For the fluorescence displacement assay, 1:1 complex of Rev1-CT with FAM-Polκ-RIR peptide (Polκ residues 560-575 modified with the N-terminal fluorescent FAM label; purchased from GenScript, USA) was prepared by mixing the unlabeled Rev1-CT with an excess of the peptide, followed by gel-filtration chromatography and exchange of the complex into 50 mM NaH2PO4, 100 mM NaCl, 10% D2O, pH 7.0 buffer. 2.3.2. Fluorescence titration assays. Disruption of the Rev1-CT/RIR PPI by analogues 4-15 was examined by fluorescence titration experiments. Fluorescence intensities of solutions of the FAMlabeled polκ-RIR complex with Rev1-CT (0.1 µM) in binding buffer were measured in the absence



and presence of various concentrations (0.1 µM - 0.5 µM) of compound following a 1 min incubation. The binding buffer (pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4. Samples (20 μl) were measured in Perkin Elmer black 396-well polypropylene assay plates. All measurements were carried out in duplicate for each specific condition, and the average fluorescence values were obtained. Fluorescence intensity measurements and data analysis were performed using a BioTek Synergy H1 hybrid reader and Gen5 microplate data collection and analysis software. The fluorescent probes were excited at 485 nm and the fluorescence intensity was detected at the emission maximum 528 nm. Keeping the FAM-labeled polκ-RIR complex with Rev1-CT (0.1 µM) concentration constant with increasing concentration of the ligand resulted in disruption of the FAM-labeled polκ from Rev1-CT, leading to an increase in fluorescence intensity associated with free FAM-labeled polκ. The area under the curve (AUC) for each individual concentration was calculated and utilized to determine the IC50 of each compound per the literature protocol32 as the concentration at which F − F0 = (F∞ − F0)/2, where F is the measured fluorescence intensity of a solution containing the FAMlabeled polκ-RIR peptide - Rev1-CT complex at a given concentration of the compound, F0 is the fluorescence intensity of a solution of the FAM-labeled polκ-RIR complex with Rev1-CT, and F∞ is the fluorescence intensity of the free FAM-labeled polκ-RIR peptide at 0.1 µM. The raw data from the fluorescence titration assays for 3 and a reference compound inhibitor (compound 5 from Reference 18) is depicted in Figure S4. 2.3.3. NMR binding experiments. Two types of NMR binding experiments were performed. In the first set of experiments, 15N labeled Rev1-CT (50 µM, 2% DMSO, 15 ºC) was titrated with increasing amounts of compounds 1 dissolved in matching buffer. The binding was monitored by recording 2D 1H-15N HSQC spectrum of Rev1-CT at each point of titration on an 18.8T Agilent


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VNMRS spectrometer (800 MHz 1H frequency) equipped with a cold probe. In the second series of experiments, compound 2 (50 µM, 0% DMSO, 20 ºC) was titrated with unlabeled Rev1-CT and monitored by collecting 1D

19F

NMR spectra of the compounds on a 9.4 T Varian Inova

spectrometer (400 MHz 1H frequency). 2.3.3.1. 1H-15N HSQC NMR titration data. Binding of the compounds to 15N-labeled Rev1-CT resulted in small chemical shift changes in 1H-15N HSQC spectra (< 0.1 ppm (1H)) and was accompanied by gradual movement for a subset of Rev1-CT peaks towards their positions in the compound bound state (fast on the NMR time scale exchange; kex >> Δ  γB0Δ, where kex is the sum of forward and reverse rates, Δ and Δ are chemical shift and frequency differences between the exchanging states, γ is gyromagnetic ratio of a nucleus, B0 is static magnetic field strength). The titration profiles (cumulative 1H and 15N chemical shifts  = (H2+(γN/γH N)2)1/2 measured as a function of protein and ligand concentrations, P0 and L0) were fit to a two-state binding model to extract dissociation constant Kd and chemical shift differences between the bound and free states Δ for the backbone and Trp side-chain NH groups. In the first step, the total chemical shift change  for all non-overlapped peaks was fit to the following equation:

   0  

L0  [ L] , P0

(1)

1 [ L]  ( ( P0  L0  K d ) 2  4 L0 K d  ( P0  L0  K d )) . 2 In the second step, the chemical sift differences between free and bound state Δ for each NH group were extracted from fits of individual titration profiles with Kd fixed to the value obtained in the first step. The obtained binding-induced NMR chemical shift changes for NH groups of Rev1-CT were mapped onto the domain structure, outlining the binding site of Rev1-CT for 1.



The analysis of NMR binding data for Rev1-CT with 1 was complicated by limited water solubility of the compound (50 µM 15N labeled Rev1-CT was titrated with 500 µM of 1). We were not able to reach saturation of Rev1-CT with the compound and analyzed the initial portion of the titration curve. At these conditions, dissociation constant Kd and chemical shift differences  extracted from the titration profiles are correlated with each other (weaker Kd correspond to higher Δ), resulting in large errors of the obtained absolute values of Δ and Kd (53 ± 44 μM for 1 was extracted from data fit). However, relative Δ obtained for different residues can be used for precise mapping of Rev1-CT binding site for the compound. 2.3.3.2. 19F NMR titration data. Taking advantage of the fact that compounds 2 and 3 contain fluorine nuclei, we studied Rev1-CT – compound binding using 19F NMR. 19F is spin-½ nucleus with high gyromagnetic ratio (0.83γH) and chemical shift range spanning ~400 ppm (ref). The only peak in 1D

19F

NMR spectrum of the 2 gradually disappeared upon titration with increasing

concentrations of Rev1-CT, while a new peak corresponding to a 2:Rev1-CT appeared ~8 ppm (19F) away from the peak of the free 2. The exchange between free and bound states of 2 is slow on the NMR time scale (kex > Δ.


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3. RESULTS AND DISCUSSION 3.1. Identification of a druggable interface on Rev1-CT Recent studies have shown that the majority of binding free energy of a PPI is primarily governed by only a few amino acid residues, which can provide druggable sites that can be targeted with small molecules33-34. To improve our understanding of the molecular recognition at the Rev1CT/RIR and Rev1-CT/Rev7/Rev3 binding interfaces and to identify potential hot spot residues for these PPIs, we analyzed key interactions between Rev1-CT and its partner proteins. In this work, previously determined three-dimensional crystal structures of human REV1 in complex with the RIR motifs from PolD3 (2N1G)15, Polη (2LSK)11, and Polκ (4GK5)35 and Rev1-CT in complex with Rev7/Rev3 (4GK0)35 were analyzed through a series of computational studies. Figure 1 shows the three-dimensional structure of the human Rev1-CT domain (residues 1157-1251) in complex with the polκ-RIR (residues 564-573) and the Rev7 subunit of polζ (residues 9-209) bound to a Rev3 peptide (residues 1874-1893). Using this complex as our reference structure, the Rev1-CT/RIR and Rev1-CT/Rev7 binding interfaces were evaluated for potential small molecule binding sites.



Figure 1: The three-dimensional crystal structure of the human Rev1-CT domain (residues 11571251) in complex with the Rev7 subunit of Polζ and the Polκ-RIR motif (PDB code: 4GK5). The secondary structure of the protein complex colored as follows: CT domain of Rev1 = blue, Polκ = yellow, Rev7 = green, Rev3 = red. The Rev1-CT/RIR binding interface = green color box; the Rev1-CT/Rev7 binding interface = dotted green color box. A key structural motif of the RIR peptide sequence is two consecutive Phenylanine (F) residues in the central portion of the motif (Figure 2) that interact with Rev1-CT through strong hydrophobic interactions. The type I′ β-hairpin of Rev1-CT, which packs tightly against helices α1 and α2, together with the hydrophobic side chains of L1171 and W1175 (α1) and the negatively charged side chain of D1186 (α2) form a prominent pocket on the surface of the domain capable of accommodating the two FF residues of RIR motifs. Calculations of the size and shape of the FF binding site on Rev1-CT performed using the CAST program36 demonstrated that it consists of 145.37 Å2 of hydrophilic surface area and 283.63 Å2 of hydrophobic surface area. The total volume of the pre-formed FF binding pocket on Rev1-CT (393 Å3) is similar to that typically associated with small molecule drug-like sites (~300 Å3)37. The surface groove leading to the binding site is lined with hydrophobic residues (L1159, V1160, L1171, L1172, and W1175) and the opening of


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the binding site primarily consists of charged residues (E1174, T1178, D1186 and Q1189) (Figure 2).

Figure 2. The binding interface of the Rev1-CT/RIR PPI. (A) The molecular surface area of the FF binding site on Rev1-CT. Red = charged negative residue; Blue = charged positive residue; Green = hydrophobic residue. (B) Enlarged view showing the binding conformations and key interactions of the Polκ RIR in complex with Rev1-CT (Green = α1; Blue = α2; Orange = α3; Red = α4). (C) Dynamic hydrogen bond (yellow dotted line) and T-shaped π-π interaction (pink dotted line) between the Polκ RIR and Rev1-CT. Several potential hot spot residues of Rev7 at the Rev1-CT/Rev7 interface have also been characterized through structural and mutational studies35,38. These Rev7 residues (L186, P188, and Y202) interact with a well-characterized hydrophobic region on Rev1-CT. CAST analysis of the Rev1-CT/Rev7 binding interface demonstrated that this PPI primarily consists of polar grooves, but not a prominent binding pocket that can serve as an anchoring point for a small molecule ligand. Because the interaction region for the Rev1-CT/Rev7 interface is flatter, induced only upon binding, and the volume of the most drug-like binding sites for this PPI is small (~10 Å3), we chose to focus our design efforts at the Rev1-CT/RIR interface.



3.2. Small molecule lead identification In order to gain a more detailed picture of the binding energetics for each individual amino acid residue involved in the Rev1-CT/RIR PPI, a molecular dynamics (MD, AMBER14)39 study of Rev1-CT in complex with the Polκ-RIR (PDB ID: 2LSI) was carried out followed by a binding free energy analysis (MM/GBSA)40. Our pair wise, per-residue free energy decomposition profiling revealed that the most favorable interactions with Rev1-CT are formed by K564, S565, F567 and F568 of the Polκ-RIR (Table 1). Not surprisingly, the contribution of F567 and F568 are particularly significant, and are dominated by favorable van der Waals and non-polar solvation terms with W1175 of Rev1-CT. These two residues are stable in the binding region and the side chain phenyl ring of F568 forms a T-shaped π-π interaction with W1175 (Figure 2). The two nitrogen atoms present at the backbone of the F567 and F568 residues demonstrate favorable dynamic intermolecular hydrogen bond (-7.77 kcal/mol and -5.30 kcal/mol, Table 1) with the side chain carbonyl of D1186. Furthermore, the adjacent amino acid residues K564 and S566 form a network of hydrogen bonds with D1186, Q1189 and D1186, respectively. Although K564 forms a tight hydrogen bond with D1186 (-31.13 kcal/mol), the polar solvation decomposition energy disfavors this specific interaction, as the electrostatic interaction between K564 and D1186 is exposed to the solvent accessible surface area of the protein. Overall, our per residue energy decomposition studies suggest that a small molecule inhibitor of the Rev1-CT/RIR PPI should demonstrate strong π-π interactions with W1175 while also having the ability to form hydrogen bonds with the polar residues present on the surface groove of Rev1-CT at the RIR interface. Based on both the conserved nature of the FF motif and its established importance as a Rev1-CT/RIR interaction hot spot, we sought to identify small molecule scaffolds that would


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structurally mimic the three-dimensional orientation of these two residues within the Rev1CT/RIR complex. First, we extracted the FF residues from the polκ-RIR and performed a ‘score in place’ docking in Glide XP mode to ensure that the isolated residues would adopt the same structural orientation as in the polκ-RIR peptide bound to the Rev1-CT domain. Analysis of the root mean square deviation (RMSD) between the full polκ-RIR and the isolated residues in complex with Rev1-CT demonstrated that both structures correlated very well when superimposed (RMSD < 0.1 Å). Table 1. Pair wise per-residue free energy decomposition of main Polκ residues with Rev1-CT.a Polκ-RIR Residue K564 S566

F567

F568

Rev1-CT Residue E1174 W1175 M1183 E1185 D1186 Q1189 L1171 E1174 W1175 I1179 D1186 L1159 A1160 L1171 W1175 D1186 Q1189 V1190

van der Waals -0.12b -0.05 -0.64 -0.71 0.72 -0.03 -0.69 -0.82 -1.73 -0.77 -0.21 -1.24 -0.79 -0.70 -1.50 -1.24 -1.26 -0.54

Electrostatica -31.13 0.26 -0.51 -1.20 -15.77 -0.20 -0.21 -0.14 -0.11 0.04 -7.77 -0.12 -0.11 0.04 -0.12 -5.30 0.39 0.17

Polar Solvationa 29.05 -0.35 0.14 1.48 2.59 -0.62 0.25 0.16 -0.18 -0.06 1.89 -0.10 -0.02 0.01 0.40 2.70 -0.70 -0.18

Non-Polar Solvationa -0.32 0.00 -0.42 -0.61 -0.59 -0.29 -0.45 -0.60 -1.21 -0.85 -0.56 -0.91 -0.73 -0.57 -0.98 -0.89 -0.89 -0.48

Totala -2.53 -0.14 -1.43 -1.05 -13.05 -1.14 -1.09 -1.42 -3.23 -1.64 -6.66 -2.37 -1.64 -1.23 -2.19 -4.73 -2.45 -1.04

aFull

descriptions of the energetic terms are explained in the supplementary information. Polκ-RIR residues that contribute more than 1 kcal/mol to the total interaction free energy are included in the table. The pairwise per-residue free energy decomposition was calculated without the conformational entropy. bAll values are kcal/mol.

Next, we utilized Glide XP to automatically generate an e-Pharmacophore for the isolated FF residues in complex with Rev1-CT (Figure 3B). The e-Pharmacophore is generated based on a ranking of important energetic terms involved in the protein-ligand interaction. In addition, this



method allows for excluded volumes around the pharmacophore that correspond to regions of space that are occupied by the protein binding site (Figure S1). Interestingly, the e-Pharmacophore for the two FF residues consisted only of the side chain phenyl rings of the FF dipeptide, which strongly suggests that the π-π and hydrophobic interactions between the phenyl rings and their binding region on Rev1-CT are the most important intermolecular interactions at the Rev1-CT/RIR interface. As both the F residues are present in the helical structure, the orientations of their aromatic planes are orthogonal to each other, with an interatomic distance of 5.31 Å from the respective centers of their aromatic symmetry. Taking into consideration the overall conformation of the FF residues generated by the e-Pharmacophore method as well as the spatial arrangement of the electrostatic interactions from the per residue MM-GBSA study, we performed an initial scaffold search in our synthetic laboratory inventory to identify structures that would closely mimic the bound RIR FF residues. Our search identified 1 [(E)-3-(2-phenyldiazenyl)pyridine-2,6-diamine, commonly known as phenazopyridine] as a close structural mimic of the bound RIR FF motif (Figure 3B and 3C). Docking of 1 at the RIR interface of Rev1-CT suggests it adopts a similar orientation as the parent FF motif. (Figure 3D). The aromatic symmetry of 1 in both its free and bound conformations are similar, suggesting a favorable free binding energy. The interatomic difference for the two phenyl rings in 1 is slightly larger than that of the polκ-RIR FF residues (6.31 Å and 5.31 Å, respectively); however, an overlay of the two structures demonstrates comparable alignment of the phenyl rings between the two scaffolds.


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(A) N N H2N

N

R' R

NH2

1; R = R' = H 2; R = F, R' = H 3; R = R' = F

Figure 3. Identification of a structural mimic of the RIR FF motif. (A) Structure of phenazopyridine and initial analogues. (B) The e-Pharmacophore generated by the extracted FF residues superimposed on the crystal structure of polκ. (C) A similar e-Pharmacophore was generated following docking (Glide XP) of 1 with Rev1-CT. The two aromatic surfaces are represented in pink sphere with inter-site distance in pink dotted line. (D) An overlay of the docked conformation of 1 (blue) on the polκ RIR demonstrates an orientation comparable to the RIR FF residues. We performed MD simulations (20 ns) followed by extensive post-dynamic analyses on the 1:Rev1-CT complex to further characterize the stability of 1 bound to Rev1-CT and explore the nature of key intermolecular interactions between 1 and specific amino acids at the Rev1-CT binding site. Compound 1 fits well inside the binding pocket of Rev1-CT and demonstrates a strong T-shaped π-π stacking interaction with W1175 (occupancy > 59%, Figure S2). The two amino groups of compound 1 interact with D1186 and E1174 through water bridges with an occupancy rate of approximately 50%. (Figure 4A). Interestingly, the ligand torsional profile for



the docked 1:Rev1-CT complex suggests a reduced stability of the phenyl ring inside the binding site, primarily resulting from the rotatable nature of the N-C bond. To stabilize the rotatable bond and improve both the van der Waals and π-π stacking interactions, we designed two analogues of 1 that incorporated fluorine atoms at either the 2- or 2,3-positions of the phenyl ring (2 and 3, respectively). Docking and subsequent MD studies of these two analogues in complex with Rev1CT suggested that the incorporation of the fluorine atoms reduces the rotatable nature of the N-C bond and results in a sandwich-type π-π stacking interaction with W1175, which improves the binding energy and stability of the ligand inside the Rev1-CT active site (Figure 4B and C). In addition, both amino moieties of the pyridine ring interact with E1174 and D1186 through a water bridge. The contribution of individual Rev1-CT amino acid residues to interactions with the scaffold was calculated through a per residue energy decomposition study. The key residues (contributing more than 1 kcal/mol to the total interaction free energies) are depicted in Figure 5. The negatively charged E1174 and D1186 residues act as ‘gatekeepers’ to the hydrophobic binding pocket described above and their primary contributions to the total interaction free energies is through electrostatic interactions with the scaffold. These contributions can be seen most prominently with D1186, which contributes approximately -2.00 kcal/mol to the Rev1-CT interaction free energy for each of the three compounds. More specifically, the carbonyl side chain of D1186 forms a water-mediated hydrogen bond with the amino group of the pyridine ring of compounds 1-3. The phenazopyridine scaffold forms nonpolar interactions with the hydrophobic residues that line the binding pocket (L1159, A1160, L1171, I1179, L1172 and W1175). Most notably, van der Waals interactions between the phenyl ring and W1175 in the hydrophobic cavity make significant favorable contributions to the total interaction free energies (> 4.0 kcal/mol,


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Figure 5). These van der Waals interactions play an essential role in controlling the orientation of the phenyl ring to maintain the π-π stacking interactions with W1175.

Figure 4. Torsonal profile and stabilization of the rotable N-C bond of 1 (A), 2 (B) and 3 (C). The addition of fluorines to the core structure stabilizes the π-π interaction between the aromatic ring and W1175. Compound 1 demonstrates T-Shape π-π stacking with W1175, while 2 and 3 demonstrate sandwich type π-π stacking with W1175.



Figure 5. Individual contributions of binding site amino acids to the free binding energies (kcal/mol ± SEM) between Rev1-CT and the phenazopyridine scaffold.

3.3. Experimental lead validation To validate our computational studies, we synthesized compounds 1-3 through standard diazotization coupling procedures and evaluated their ability to disrupt the Rev1-CT/RIR PPI (Scheme 1). Of note, the auto-fluorescent nature of the phenazopyridine scaffold prevented us from using the fluorescence polarization assay we previously optimized to identify inhibitors of this PPI18. For this reason, we developed a related assay that measures the increase in total fluorescence intensity associated with free FAM-polκ-RIR following its disruption from the FAMpolκ-RIR/Rev1-CT complex when incubated with an inhibitor of the PPI. An extensive analysis of the fluorescent properties of 1-3, the free FAM-polκ-RIR, and the FAM-polκ-RIR/Rev1-CT complex, provided a range of emission wavelengths for free FAM-polκ-RIR that does not overlap


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with our analogues (Figure S4). During the assay, the concentration of the small molecule is increased until the AUC becomes saturated. Interestingly, for several compounds we saw a decrease in the free FAM-polκ-RIR signal when the compound concentration reached approximately 1-2 µM. It is unclear whether this decrease is due to FRET between the compound and free FAM-polκ-RIR or if the fluorescent signal is quenched in the presence of these specific compounds. Nonetheless, AUC values that correspond to displacement of >85% FAM-polκ-RIR from the complex were obtained for all compounds allowing for the calculation of an IC50 value through the equation described in the Methods section. Through this assay, compounds 1-3 were all validated as inhibitors of the Rev1-CT/RIR PPI (Table 2, Figure S5). In addition, the results from this assay supported our design strategy and computational binding analysis, i.e. incorporation of the fluorine atoms on the phenyl ring resulted in an increase in disruption of the PPI [IC50 values = 0.99 μM (1), 0.87 μM (2) and 0.39 μM (3)].

R

N N+

NH2 F

NaNO2 HCl / H2O

F

H2N

N

N N

NH2

H2O/CH3COONa

H2N

N

NH2

1: R = H 2: R = 2-F 3: R = 2,3-F

Scheme 1. Synthetic route for compounds 1-3. To explore direct binding interactions between our lead scaffold and Rev1-CT, we analyzed chemical shift perturbations (CSPs) in the 1H-15N HSQC spectra of 15N-labeled Rev1CT upon titration of increasing concentrations of compound 1. NMR binding studies with 1 resulted in CSPs for several amino acid residues located in the FF binding site on Rev1-CT (Figure 6A and 6B), providing experimental evidence of the compound binding to the Rev1-CT/RIR interface; however, due to the low aqueous solubility of 1 at the concentrations needed for these



studies, we were unable to saturate Rev1-CT with the compound and obtain accurate estimates of dissociation constants (Kd) and chemical shift differences between the free and bound states (. We also took advantage of the fluorine present in 2 to confirm its interaction with Rev1-CT by 19F NMR. The single peak visible in the 19F NMR spectra corresponding to free compound 2 decreased following titration with increasing concentrations of Rev1-CT, while a second peak corresponding to a Rev1-CT:2 complex appeared ~8 ppm away and increased with increasing amount of added Rev1-CT (Figure 6C). Taken together, these results clearly demonstrate that the phenazopyridine scaffold disrupts the Rev1-CT/RIR PPI through direct binding interactions with Rev1-CT.

Figure 6. The PAP scaffold binds to Rev1-CT. (A) Selected regions of 1H-15N HSQC spectrum of Rev1-CT in the course of its titration with compound 1 (red to blue gradient corresponds to 0:1 to 2.5:1 1:Rev1-CT ratio). (B) Cumulative 1H and 15N CSPs induced by compound 1 binding mapped onto the Rev1-CT structure. (C) 19F spectrum of 2 (50 µM) upon its titration with increasing amounts of unlabeled Rev1-CT.


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3.4. Lead Optimization Based on the promising properties of lead analogues 1-3, we sought to further modify and improve their TLS inhibitory activity through the addition of glycine and alanine residues appended to the pyridine amines (Scheme 2). We chose to append these amino acid residues at the 2- and 6-positions for two main reasons. First, as our modeling predicts that the pyridine moiety orients towards the solvent accessible surface of Rev1-CT, the addition of small, hydrophobic amino acids would reduce the polar interactions between the pyridine amines and water molecule present at the surface of the pocket. Second, our modeling suggested that the addition of these residues could improve intermolecular hydrogen bonding interactions between the scaffold and Rev1-CT; most notably, between the amino group of the phenazopyridine and D1186. With this in mind, we synthesized and evaluated a series of derivatives of 1-3 that incorporate a glycine or alanine residue at either the 2- or 6-position of the pyridine moiety (Table 2, 4-15).

N-Boc glycine/ N-Boc alanine DMAP, EDC N

H2N

DCM, 36hr

NH2

O

H N

O

N H

O

I

N

O

H N

O

NH2

N H

O

II

N

NH2

III

NH2 HCl / NaNO2 / H2O

R

R

R R

R N N H2N

N

O

N H

N N H N

O

H2N

N N

O N

H N

N H

O IIa: R = H IIb: R = 2-F IIc: R = 2,3-F

O

H2N

N

O

H N

N H

O IId: R = H IIe: R = 2-F IIf: R = 2,3-F

N N

O

H2N

N

N H

4: R = H 5: R = 2-F 6: R = 2,3-F

N H

H N

O

O IIIj: R = H IIIk: R = 2-F IIIl: R = 2,3-F R

R N N

NH2

N

IIIg: R = H IIIh: R = 2-F IIIi: R = 2,3-F

R O

H2N

DCM, TFA

R N N

O

O

H2N

N N

O N

NH2

N H

7: R = H 8: R = 2-F 9: R = 2,3-F

H2N

N

N N

O

N H

NH2

10: R = H 11: R = 2-F 12: R = 2,3-F

Scheme 2. Synthetic route for compounds 4-15.


H2N

O N

N H

13: R = H 14: R = 2-F 15: R = 2,3-F

NH2


The addition of a glycine residue to either position (4-9) had minimal effects on the ability of the lead scaffold to disrupt the Rev1-CT/RIR PPI. The one exception to this was modification of the core structure of 2 by the addition of a glycine at the 6-position (8), which significantly enhanced the ability of the parent scaffold to disrupt the PPI (IC50 values = 0.87 and 0.51, respectively). Our modeling for analogue 8 shows that the 2-fluoro phenyl ring penetrates deeply into the binding pocket on Rev1-CT and forms a sandwich type π-π interaction with W1175, while the side chain amine forms a dynamic hydrogen bond with E1174 and the carbonyl group present at the backbone of W1175 (Figure 7A). In addition, incorporation of the glycine allows the free amine at the 6-position to interact with D1186 through a water bridge. The addition of an alanine residue at the 2-position had no impact on the ability of the compounds to disrupt the Rev1-CT/RIR PPI. The IC50 values for each of these analogues (10-12) were comparable to the unsubstituted compounds (1-3). For alanine substitutions on the 6-position, a significant decrease in activity was seen for the 2,3-difluoro substituted analogue (15). We performed additional computational studies to explore which potential interactions account for the discrepancy in activity seen with the 2,3-difluoro substituted compounds. Comparing analogues 6 and 12 (2-substituted glycine and alanine, respectively) suggests that the Cα methyl group on the alanine side chain orients toward the solvent accessible surface, which positions the protonated amine group towards D1178, resulting in strong dynamic hydrogen bonds with the side chain carbonyl of D1178 (Figure 7B). By contrast, the protonated amine group of the glycine is exposed to the solvent accessible surface, reducing specific interactions with Rev1-CT. The addition of a glycine (9) or alanine (15) to the 6-position shifted the phenyl ring of the compound in the binding pocket to prevent deep penetration of the difluorophenyl ring and reduced the ability of either compound to maintain a constant hydrogen bond with E1174 (Figure S3). The pair wise per-


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residue free energy decomposition for all our compounds (1-15) with Rev1-CT are described in Supplementary Table 1.

Table 2. Inhibitory activity of phenazopyridine analogues. N N R3HN

N

R1

NHR2

Compound

R1

R2

R3

IC50 (μM)a,b

ΔG (Kcal/mole-1)c

1

H

H

H

0.99 ± 0.3

-4.63

2

2-F

H

H

0.87 ± 0.6

-4.27

3

2,3-F

H

H

0.39 ± 0.2

-5.69

4

H

glycine

H

1.02 ± 0.7

-4.45

5

2-F

glycine

H

1.62 ± 0.5

-4.50

6

2,3-F

glycine

H

0.81 ± 0.5

-5.50

7

H

H

glycine

1.07 ± 0.2

-3.99

8

2-F

H

glycine

0.51 ± 0.4

-6.59

9

2,3-F

H

glycine

1.44 ± 0.9

-3.68

10

H

alanine

H

0.84 ± 0.6

-5.52

11

2-F

alanine

H

0.60 ± 0.4

-5.69

12

2,3-F

alanine

H

0.42 ± 0.3

-5.90

13

H

H

alanine

0.79 ± 0.4

-4.49

14

2F

H

alanine

0.74 ± 0.5

-5.19

15

2,3-F

H

alanine

1.47 ± 0.7

-3.98

aIC values represent the Mean ± SEM of at least two separate experiments performed in duplicate (7, 10-11, 13-15) 50 or triplicate (1-6, 8-9, 12). bAll analogues evaluated after 1-10 min of incubation time. CΔG calculated by umbrella sampling method described below.



Figure 7. Torsonal profile and stabilization of N-C rotable bond of compounds 8 (A) and 12 (B). The π-π stacking interactions play a significant role in the Rev1-CT/RIR PPI. Additions of an amino acid side chain and fluorine to the core structure enhance the stabilization of the π-π interactions between the aromatic ring and W1175. Compounds 8 and 12 both show sandwich type π-π stacking with W1175. 3.5. Absolute free binding energy calculations A key goal for continuing to develop improved inhibitors based on our lead phenazopyridine scaffold is the ability to predict whether an analogue can disrupt the PPI based on a computational binding energy between the analogue and Rev1-CT. Initially, we utilized standard MM/GBSA and MM//PBSA energy calculations for this purpose. This method uses molecular mechanical energy along with the continuum solvent approach to calculate enthalpy and entropy upon ligand/protein binding. Unfortunately, using this method provided binding free energy values that did not correlate with the results from our fluorescence displacement assay (data not shown). Possible


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reasons for poor correlation include: (1) an inaccurate calculation of the conformational entropy of the protein and ligand structure, (2) MM/PBSA and MM/GBSA calculations do not account for structural changes in the receptor and ligand upon binding, or (3) absolute free energy is difficult to calculate in a finite length and time scale simulation. With this in mind, we adopted an umbrella sampling approach30, which extracts the binding energy of a ligand from the potential mean force (PMF) of overlapping conformations of the protein-ligand complex. In our umbrella sampling study, a force constant of 1000 kJ mol−1 nm−2 and a pull rate of 0.003 nm ps−1 was applied to drive the ligand from one thermodynamic state to another. These thermodynamic states are reaction coordinates or sampling windows of a system that can be defined as the distance (ξ, nm) between the center of mass of the ligand molecule and the center of mass of the protein. Intermediate steps in this process are covered by a series of reaction coordinates or sampling windows where molecular dynamics simulation and free energy of each reaction coordinate is calculated. All of the intermediate reaction coordinates are combined and the free binding energy landscape (PMF curve) as a function of the distance between the ligand center of mass and Rev1-CT is determined by the weighted histogram analysis method (WHAM)41. The ΔG value for each compound is the energy minimum of its respective PMF curve (Table 2) and each free binding energy calculated from the umbrella sampling method correlated well (r2 = 69%) with the experimentally determined logIC50 values demonstrating that this is a valid method to predict whether small molecules will disrupt the Rev1CT/RIR PPI (Figure S8). The calculated PMF curves for compounds 1-3 as a function of the distance between the ligand center of mass relative to Rev1-CT is depicted in Figure 8. Not surprisingly, the energy minimum for each of these compounds was seen when each was the same distance from Rev1-CT (0.83 nm)



For compounds 1 and 3, the free energy rises slowly following the sharp energy minimum at 0.83 nm, crossing zero at 1.19 nm, and reaching a plateau at 1.24 nm. This plateau denotes the distance between the compound and Rev1-CT at which no interaction between the two occurs. Interestingly, the PMF curve for compound 2 shows a narrow energy trough for a metastable complex at 0.83 nm, followed by an energy minimum at 1.24 nm. The two energy troughs for compound 2 are most likely a result of two stable conformations resulting from rotation of the CN bond within the binding site of Rev1-CT as described above.

Figure 8. PMF curves for compounds 1-3 calculated through umbrella sampling. 4. CONCLUSION TLS has emerged as a promising therapeutic target to complement first-line genotoxic chemotherapeutics such as cisplatin and cyclophosphamide17,42. Inhibitors of TLS can increase the efficacy of these agents in human cancer while also reducing the acquired resistance associated with these drugs. We have demonstrated previously that disruption of the Rev1-CT/RIR PPI inhibits TLS in human cancer cells, presumably by disrupting the scaffolding function of Rev1


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within the hetereoprotein complex that governs Rev1/Polζ-dependent TLS. We previously developed and optimized a screening assay to identify small molecules that disrupt this PPI. To expand the chemotypes that bind to Rev1-CT and disrupt TLS, we have utilized a structure-based computational approach to identify the phenazopyridine scaffold that mimics the FF hot spot residues present in all RIR motifs. This scaffold disrupts the Rev1-CT/RIR through direct binding at the RIR interface on Rev1-CT. Follow-up SAR and computational modeling studies provide key information as to regions of the scaffold that can be modified to improve activity while also validating umbrella sampling as a method for correlating computationally-derived binding free energy with activity in biological binding or disruption assay. The further development of this scaffold for its novel anti-cancer properties is ongoing.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Supporting Information includes compound synthesis protocols and final analogue characterization.

AUTHOR INFORMATION Corresponding Author *Tel: 1-860-486-8446. Fax: 1-860-486-6857. Email: [email protected]

ORCID Kyle Hadden: 0000-0001-9482-1712



ACKNOWLEDGEMENTS The authors would like to thank the University of Connecticut Research Foundation and the Connecticut Institute for Clinical and Translational Sciences for funding. The authors would like to thank the High Performance Computing (HPC) facilities at the University of Connecticut.

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Graphical Abstract


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Structural Approach To Identify a Lead Scaffold That Targets the

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