Ligand Proton Pseudocontact Shifts Determined from Paramagnetic

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Ligand Proton Pseudocontact Shifts Determined from Paramagnetic Relaxation Dispersion in the Limit of NMR Intermediate Exchange Difei Xu, Bin Li, Jia Gao, Zhijun Liu, Xiaogang Niu, Gilbert Nshogoza, Jiahai Zhang, Jihui Wu, Xun-Cheng Su, Wei He, Rongsheng Ma, Daiwen Yang, and Ke Ruan J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b01443 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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The Journal of Physical Chemistry Letters

Ligand Proton Pseudocontact Shifts Determined from Paramagnetic Relaxation Dispersion in the Limit of NMR Intermediate Exchange ⊥

Difei Xu, † Bin Li, ‡ Jia Gao, †, Zhijun Liu, # Xiaogang Niu, § Gilbert Nshogoza, † Jiahai Zhang, † Jihui Wu, † Xun-Cheng Su, ∥ Wei He, ‡ Rongsheng Ma, *, † Daiwen Yang *,▽ and Ke Ruan *,† †

Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences,

University of Science and Technology of China, Hefei, Anhui, 230027, P. R. China ‡

Department of pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua-

Peking Joint Center for Life Sciences, Tsinghua University, Beijing, 100084, P. R. China ⊥

Center of Medical Physics and Technology, Hefei Institute of Physical Science, Cancer

Hospital Chinese Academy of Science, Hefei, Anhui, 230031, P. R. China #

National Facility for Protein Science in Shanghai, ZhangJiang Lab, Shanghai Advanced

Research Institute, Chinese Academy of Sciences, Shanghai, 201210, P. R. China §

Beijing Nuclear Magnetic Resonance Center, College of Chemistry and Molecular Engineering,

Peking University, Beijing 100871, P. R. China

ACS Paragon Plus Environment

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State Key Laboratory of Elemento-Organic Chemistry, Collatorative Innovation Center of

Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, P. R. China ▽

Department of Biological Sciences, National University of Singapore, Singapore, 117543,

Singapore AUTHOR INFORMATION Co-first Author Difei Xu, Bin Li and Jia Gao. Corresponding Author *Email:

[email protected]

(R.M.).

*Email:

[email protected]

(D.Y.).

*Email:

[email protected] (K.R.).

Delineation of protein-ligand interaction modes is key for rational drug discovery. The availability of complex crystal structures is often limited by the aqueous solubility of the compounds, while lead-like compounds with micromolar affinities normally fall into the NMR intermediate exchange regime, in which severe line broadening to beyond the detection of interfacial resonances limits NMR applications. Here, we developed a new method to retrieve low-populated bound-state 1H pseudocontact shifts (PCSs) using paramagnetic relaxation dispersion (RD). We evaluated using a 1H PCS-RD approach in a BRM bromodomain lead-like inhibitor to filter molecular docking poses using multiple intermolecular structural restraints. Considering the universal presence of proton atoms in drug-like compounds, our work will have wide application in structure-guided drug discovery even under an extreme condition of NMR intermediate exchange and low aqueous solubility of ligands.


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TOC GRAPHICS

KEYWORDS: NMR spectroscopy, relaxation dispersion, intermediate exchange, bromodomain inhibitor, pseudocontact shift


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Understanding the molecular basis of protein-ligand interactions is central to rational drug discovery. It is intuitive to analyze protein-ligand interaction modes in high-resolution complex crystal structures1, which are, however, not always available due to the low aqueous solubility of compounds and/or dynamic nature of targets. The protein-ligand interaction modes have been alternatively characterized by a variety of nuclear magnetic resonance (NMR) techniques that enrich valuable structural restraints, e.g., chemical shift perturbation2, intermolecular nuclear Overhauser

effect

(NOE)3-4,

transferred

paramagnetic

relaxation

enhancement5-7

or

pseudocontact shifts (PCSs)8-14. These methods are, however, not feasible in the NMR limit of intermediate exchange regime, with severe line broadening to beyond the detection of interfacial resonances15. Lead-like compounds with affinities of approximately 10 µM to 10 nM often have the exchange rates approximate the chemical shift deviations between the free and bound states. These leads thus burst in the undesirable intermediate exchange regime, in which it remains difficult to depict a protein-lead interaction mode despite its essential role in structure-guided lead optimization. We have recently demonstrated that the line broadening effect in the intermediate exchange regime can be alleviated by a highly skewed protein-ligand molar ratio, in which low-populated bound-state

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F PCSs were retrieved from paramagnetic chemical exchange saturation transfer

(CEST)16-17. Because of the ubiquity of proton atoms in chemical compounds, it is highly desirable to develop a new approach to extract 1H-observed intermolecular restraints within the NMR limit of intermediate exchange. Since 1H CEST suffers from 1H-1H NOE interference18-20, relaxation dispersion (RD)21-23 could be used to extract the ligand 1H chemical shifts of lowpopulated bound states in the intermediate to slow exchange time scale, or ligand 1H PCSs if the target protein is labeled by a lanthanide ion with a paramagnetic susceptibility tensor. RD has


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been extensively applied to isolated nuclei without homonuclear scalar couplings24-26. The undesirable evolution of proton homonuclear scalar couplings can be repressed by a selective refocusing of the magnetization of the proton of interest in the middle of the RD period27; however, it is not feasible to selectively refocus the widely distributed ligand 1H signals. Alternatively, the “perfect echo” scheme has been applied to suppress the J modulation in CarrPurcell-Meiboom-Gill (CPMG) experiments to measure the transverse relaxation rate of small molecules28. The ligand-observed RD experiment for singlet 1H signals has been introduced for extracting binding kinetics of lead-like compounds to a target29. Here, we encoded the “perfect echo” element to suppress the J modulation in 1H RD, which allows the determination of the low-populated bound-state ligand 1H PCSs (we termed this method 1H PCS-RD). These valuable intermolecular PCS restraints facilitated the identification of best-fit docking poses even at the limit of the NMR intermediate exchange. During our fragment-based lead discovery campaign30-31 against the BRM bromodomain associated with a variety of diseases32-34, we identified some small molecule inhibitors, including the micromolar affinity compound 1 (Figure 1a). Upon titration of 1 to the

15

N labeled BRM

bromodomain, binding-site resonances, such as L43, S45, V60, F62, K63, disappeared (Figure 1b), which is a typical phenomenon of NMR intermediate exchange. Conversely, a quite dispersed 1H spectra of this inhibitor was observed (Figure 1c) even in the presence of the BRM bromodomain at a low concentration (4%, molar ratio). The aromatic proton signals were assigned using the 2D 1H-13C HSQC, 1H-13C HMBC and 1H-1H TOCSY spectra (Figure S1). The ligand 1H signals became indistinguishable upon titration of 50% (molar ratio) of the BRM bromodomain due to the severe line broadening in the NMR intermediate exchange and interference from the protein proton signals (Figure 1c). That is to say, the ligand-observed 1H


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spectra and accompanying structural restraints have to be acquired at a very low protein concentration. To suppress artifacts from homonuclear scalar couplings, the “perfect echo” element (τ-180-τ90-τ-180-τ module, τ is the delay between pulses) was incorporated into the ligand-observed 1H RD pulse scheme (Figure S2). To assess its performance on coupled homonuclear systems, we first performed simulations on two-spin and three-spin systems without chemical exchange. That is, a flat RD profile was expected if the homonuclear scalar couplings could be well suppressed. Our simulation results indicated that the offset effect was negligible when the carrier frequency offsets were smaller than 800 Hz. The pulse imperfection effect could also be neglected when the pulse error was smaller than 4% (Figure S3 and S4). To reduce the offset and pulse imperfection effects, it is necessary to set the carrier at the center of the signals of interest and calibrate the pulse width carefully, which is the same as the guideline for CPMG RD35-36. Multiple RD profiles could be in principle acquired with varying carrier offsets in the case of a large distribution range of 1H chemical shifts. When the CPMG field strength υCPMG, defined as 1/4τ, is smaller than 30 Hz, the simulated relaxation rates are significantly larger than the true values for 3-spin systems (Figure S4), suggesting that the “perfect echo” scheme is not suitable for probing extremely slow chemical exchanges.


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Figure 1. NMR intermediate exchange for the BRM bromodomain inhibitor. a) Chemical structure of compound 1. b) Disappearance of the interfacial resonances of the BRM bromodomain upon titration of 1 at the annotated ligand/protein molar ratio. c) Line broadening effect of the ligand 1H spectra induced by the addition of the BRM bromodomain. Annotated are protein/ligand molar ratios.

We further synthesized the RD profiles of single-spin and two-spin systems with chemical exchanges between two states. The RD profiles of the single-spin system were slightly different when using the standard CPMG echo and the “perfect echo” element (Figure S5). Due to the difference caused by the extra 90° pulse, the RD profiles obtained by the latter should be analyzed using the modified Bloch equation (Supporting Methods) rather than the Carver & Richards equation. The extra 90° pulse also results in interconversion (or mixing) of magnetizations of spins I and S in a two-spin system. Nevertheless, the synthetic RD profiles of the two spins with varying exchange parameters could be fitted well using the modified Bloch


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equation (Figure 2). It is of particular interest that the best-fitted chemical shift difference between the free and bound states (|∆ω|) agreed reasonably well with the synthetic input, especially for the spin with a larger value of |∆ω| in the two-spin system (Table S1). Our simulation results suggest that the ligand-observed 1H RD encoding “perfect echo” element can be used to extract the chemical shifts and PCSs of low-populated bound states. We, hence, acquired the ligand 1H RD spectra encoding the “perfect echo” in the presence of an approximately 3% (molar ratio) Y3+ or Tm3+ labeled BRM bromodomain K64C mutant. This mutant and the chelation of lanthanide ions were prepared as described previously16. The total RD time was set to 60 ms at varying υCPMG from 66.5 to 666.5 Hz. The reference spectrum of the same sample was acquired without the RD module. For each 1H signal, the intensities in the reference spectrum and in the RD spectrum at a certain CPMG frequency allowed the calculation ௘௙௙

of ܴଶ , which was modulated by υCPMG if chemical exchanges occurred. As a control, four isolated 1H signals (atoms 6, 23, 25, 26) showed almost flat lines in the 1H RD profiles of the free-form compound 1 (Figure S6), indicating that there was no chemical exchange in the absence of protein. Conversely, small amounts of diamagnetic or paramagnetic-labeled proteins induced different magnitudes of curvature changes of the 1H RD profiles (Figure 3) acquired using Agilent 700 MHz and 500 MHz spectrometers, respectively.


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Figure 2. Synthetic RD profiles of a two-spin system (I and S) with JIS = 0 (a) and JIS = 8 Hz (b). The RD profiles of spin I (black) and S (red) at 700 MHz (solid circles) and 500 MHz (open circles) were best-fitted using a two-state model (solid lines). The exchange parameters and the best-fitted ones are listed in Table S1.

RD profiles of the four ligand protons acquired in two magnetic fields were then best-fitted globally using the two-state model assuming the shared exchange rate and bound-state population. Two-fold ambiguity in the bound-state chemical shift existed as the absolute value |∆ω| was retrieved for a proton resonance in the presence of the diamagnetic-labeled BRM bromodomain. Similarly, the paramagnetic bound-state chemical shift was also extracted with


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two-fold degeneracy (Figure 3). Hence, the difference between the paramagnetic and diamagnetic bound-state chemical shifts was in deed the experimental 1H bound-state PCS, but with four-fold ambiguity37.

Figure 3. Experimental 1H RD profiles of the ligand protons in the presence of approximately 3% Y3+ labeled (left)- or Tm3+ labeled (right)-BRM bromodomains. Data were acquired with 700


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MHz (red) and 500 MHz (black) spectrometers, respectively. Error bars represent 3σ. The bestfitted values of |∆ω| determined using a two-state model are annotated.

Taken into account of the four-fold ambiguity of the ligand bound-state PCSs and the degree of freedom in the ligand location and conformation, it is not feasible to directly define the protein-ligand interaction mode using 35 protein-observed 15N PCSs and four intermolecular 1H PCSs. These ligand bound-state PCSs were rather valuable structural restraints to identify the best-fitting one out of a number of docking poses. Hence, the protein-observed

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N PCSs were

used to accurately determine the lanthanide coordinates and associated paramagnetic susceptibility tensor16, which in turn allowed back-calculation of the ligand bound-state 1H PCSs of each possible pose predicted by the Autodock 4 program38. PCS fitting was based on the freeform crystal structure (PDB: 4QY4) using the Numbat program39. These docking poses with high structural similarity were divided into 15 clusters sorted by the docking energy (Figure 4a and S7). Since the conformations within the same cluster were structurally similar to each other, the lowest-energy pose was used in the following back-calculation. The experimental PCS with the closest value to the back-calculated one was selected, and their agreement was evaluated according to the Q-value40 (Figure 4b and S8). Here, the Q-value was defined as a square deviation between the experimental and back-calculated PCSs, normalized by the backcalculated PCSs. Four possible poses had a Q-value from 0.102 to 0.266, while the rest of the poses were unlikely as their Q-values were all above 0.7. The ambiguity of the poses may be ascribed to the limited number of structural restraints and/or the four-fold ambiguity of the experimental bound-state PCSs. Despite the ambiguities in the poses filtered by the 1H PCS-RD approach, the fluorophenol group protruded into the acetyl-lysine binding pocket of the BRM


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bromodomain, which was consistent with the conformation of a soluble analog revealed by the complex crystal structure16 (Figure S9).


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Figure 4. Poses consistent with the ligand bound-state PCSs retrieved from the 1H PCS-RD approach. (a) The possible docking poses of compound 1. The lowest-energy pose in each cluster is drawn in sticks while the others in lines. Red balls denote the coordinates of the lanthanide ion. (b) Correlation between the experimental PCSs (solid circles) with 3σ error bars and those back-calculated from the lowest-energy docking pose in each cluster. Four-fold ambiguity presents in experimental PCSs due to the absolute values retrieved from paramagnetic and diamagnetic RD profiles. The red solid circles represent the experimental PCS with the closest value to the back-calculated one, which was selected to calculate the Q-values. Drawn are the red dashed diagonal lines.

In summary, we developed a new approach to delineate protein-ligand interaction modes, even at the limit of the NMR intermediate exchange. As a proof-of-concept, this 1H PCS-RD method enabled the determination of low-populated ligand bound-state 1H PCSs. This method is expected to be used in applications for the depiction of protein-ligand interaction modes due to the enriched proton signals in lead-like compounds. The possible poses could, in principle, be narrowed down by additional PCS restraints, e.g., the ligand 19F PCSs or more isolated signals in the 2D 1H-13C HSQC type of RD experiments. Furthermore, multiple datasets of ligand boundstate PCSs could be collected with various protein mutants chelating with lanthanide ions. Actually, multiple PCS or residual dipolar couplings datasets have been applied to probe protein structure and dynamics40-42. This method could also be used to interrogate protein conformational dynamics using the “perfect echo” encoded RD of amide 1H or

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C with less

interference of homonuclear 1H-1H or 13C-13C scalar couplings. Our work fills the gap of NMR characterization of protein-ligand interaction modes under the extreme conditions of NMR


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intermediate exchange and a low aqueous solubility of the ligand. This structural characterization is essential for rational drug discovery from a hit to an optimized lead compound.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Synthesis, simulations, NMR experiment details and supporting table/figures are listed in Supporting Information file. (PDF) AUTHOR INFORMATION Notes DX, JG and RM: experiment and data analysis. BL and WH: synthesis. ZL, XN, GN, JZ, JW and XS: resources. RM, DY and KR: conceptualization and writing. JW, XS, WH, DW and KR: supervision. DX, BL and JG contributed equally to this work. The authors declare no competing financial interests. ACKNOWLEDGMENT Part of the NMR experiments were performed at the National Center for Protein Sciences Shanghai (NCPSS), High Magnetic Field Laboratory Chinese Academy of Sciences and the Beijing NMR Center and the NMR facility of the National Center for Protein Sciences at Peking University. J.W. and K.R. are financially supported by a grant from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08030302 and XDA12020355, respectively). K.R. is financially supported by grants from the Ministry of Science and


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Technology of China (2016YFA0500700 and 2014CB910600). K.R. and J.G. are financially supported by grants from the National Natural Science Foundation of China (U1632153 and 21703254, respectively). D.Y. is financially supported by a grant from the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2017-T2-1-125). R.M. and K.R. are financially supported by the Fundamental Research Funds for the Central Universities (WK2070080002 and WK2060190086, respectively).

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Ligand Proton Pseudocontact Shifts Determined from Paramagnetic

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