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Letter
Reviving B-Factors: Retrospective Normalized B-Factor Analysis of C-ros Oncogene 1 Receptor Tyrosine Kinase and Anaplastic Lymphoma Kinase L1196M with Crizotinib and Lorlatinib Rebecca A. Gallego, Ted W Johnson, Alexei Brooun, Daniel Gehlhaar, and Michele A McTigue ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00147 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018
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ACS Medicinal Chemistry Letters
Reviving B-Factors: Retrospective Normalized B-Factor Analysis of c-ros Oncogene 1 Receptor Tyrosine Kinase and Anaplastic Lymphoma Kinase L1196M with Crizotinib and Lorlatinib Ted W. Johnson,‡,a,* Rebecca A. Gallego,‡,a,*Alexei Brooun,a Dan Gehlhaar,a and Michele McTiguea a
Oncology Medicinal Sciences, Pfizer Inc., 10770 Science Center Drive, San Diego, CA 92121, United States
KEYWORDS normalized B-factor, temperature factor, ALK, ROS1, L1196M, crizotinib, lorlatinib ABSTRACT: Structure-based drug design (SBDD) is commonly leveraged in rational drug design. Usually, ligand and binding site atomic coordinates from crystallographic data are exploited to optimize potency and selectivity. In addition to traditional, static views of proteins and ligands, we propose using normalized B-factors to study protein dynamics as a part of the drug optimization process. A retrospective case study of crizotinib and lorlatinib bound to both c-ros oncogene kinase (ROS1) and anaplastic lymphoma kinase (ALK) L1196M related normalized B-factors to differences in binding affinity. This analysis showed that ligand binding can have protein-stabilizing affects that start near the ligand, but propagate through nearby residues and structural waters to more distal motifs. The potential opportunities for analyzing normalized B-factors in SBDD are also discussed.
Proteins are flexible and highly dynamic with motions ranging from rapid vibrations on the time scale of picoseconds and a magnitude of under 0.5 Å to conformational changes that take up to 103 seconds and sweep out greater than 10 Å.1 X-ray analysis of protein crystal structures with and without bound ligands has been a critical tool in structure-based drug design (SBDD). While there has been much work on understanding the conformation of the amino acid residues and secondary structure of proteins,1-4 less work has focused on the use of protein dynamics in SBDD. B-factors, also known as temperature factors or atomic displacement parameters, are calculated parameters in x-ray crystal structures that reflect disorder of atoms from their ideal equilibrium positions due to both thermal motion and positional disorder. Crystallographic studies done at varying temperatures have shown that dynamics have a much larger effect on atomic B-factors in general than does the crystal lattice disorder.2 In fact, typical protein crystals are composed of 40-60% solvent as measured by volume. B-factors provide insight into the dynamic motion of proteins in the crystalline state, which can be used as a model for motion in the solution state. Normalized B-factor measurements are utilized to compare motion across multiple structures. Normalized Bfactor (B') is an expression of the B-factor (B) in units of standard deviation (σB) about the mean value (µB, Equation 1). This value is calculated across all heavy atoms in a proteinligand complex crystal structure.
B' = (Equation 1)
(B
–
µB)/σB
Interestingly, while B-factors show a very large spread across high resolution co-crystal structures, normalized Bfactors show a more narrow distribution.4 Normalized B-factor has been used as a metric for analysis of protein structures to
examine motion in various sections of the protein, such as secondary structures vs. random coils, active site vs. nonactive site and buried vs. solvent-front residues.1,4 More recently, normalized B-factor analysis has been used to link specific clinically relevant anaplastic lymphoma kinase point mutations to structural disorder that correlates with constitutive activity.5 While there are many published examples that make use of normalized B-factor analyses,6-10 to our knowledge there are no known examples of use cases associated with structure-based drug design. The use of normalized B-factor is compelling in the study of ligandprotein dynamics because it constitutes an experimental measure of dynamics that validates in silico calculations such as molecular dynamics simulations. It is also complementary to other experimental methods such as hydrogen-deuterium exchange mass spectrometry (HDX)11 and nuclear magnetic resonance (NMR) experiments. While all three are highly valuable tools to investigate the motion of ligand-protein complexes, normalized B-factor analysis is uniquely capable of achieving atomic resolution. In most cases, the interaction of a reversibly bound ligand with a protein induces stabilization or “cooling” of the protein/ligand complex in rough correlation with ligand potency.12 Ligands may impact the stabilization of proximal and distal residues. These differences in stabilization, as measured by normalized B-factor, can be exploited in drug design as part of the ligand optimization process, particularly through analysis of specific changes to binding site residues. As a proof of concept for using normalized B-factor analysis to explain differences in potency, we retrospectively examined binding of first and second generation Anaplastic Lymphoma Kinase (ALK) and C-ros Oncogene 1 Kinase (ROS1) inhibitors crizotinib and lorlatinib (Figure 1). Clinically, ALK and ROS1 oncogenic translocations define a subset of nonsmall cell lung cancers (NSCLC).13-16 In addition, both
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proteins show mutations in the kinase domain as an aquired resistance mechanism to ALK or ROS1 inhibitor therapy. In the case of ALK, gatekeeper muations (ALK-L1196M) predominate.17-18 NH
O
F
Cl
N N
F Cl
H 2N
N N
O
N
O
N
N
H 2N
1 Crizotinib
N
2 Lorlatinib
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considering ligand and protein conformation. In order to complete a ligand binding analysis based on dynamics, we exploited normalized B-factors calculated from these cocrystal structures. While relatively homogeneous in global conformation, there are a few notable differences between ROS1 bound with crizotinib and lorlatinib (Figure 2a).1 Most apparently, the glycine rich loop (P-loop) and activation loop (A-loop) are unresolved in the structure with crizotinib. This implies increased motion in these non-resolved regions for the structure of ROS1 with crizotinib. Examination of the cocrystal structures colored by normalized B-factor shows that there is cooling of the residues leading to and following the Ploop for ROS1 bound to lorlatinib (Figure 2b). Furthermore, the same effect is seen in the residues comprising the A-loop.
Figure 1. Structures of crizotinib (1) and lorlatinib (2)
Crizotinib was approved by the US food and drug administration (FDA) for the treatment of ALK-positive lung cancer in 2011.19-20 While it was originally developed as an inhibitor of mesenchymal-epithelial transition factor (c-MET), it is also potent against oncogenic drivers ALK and ROS1. Crizotinib had a biochemical activity against ROS1 of 0.6 nM and a corresponding cellular IC50 of 51 nM (Table 1). Lorlatinib was developed as a next generation ALK/ROS1 inhibitor and is currently in Phase III clinical trials. Lorlatinib improved central nervous system (CNS) exposure and broad spectrum mutated ALK/ROS1 inhibition.21-22 Lorlatinib achieves biochemical ROS1 activity of 24-fold more potent than crizotinib. Furthermore, lorlatinib has cellular ROS1 IC50 values of 0.19 nM and 0.23 nM in ROS1-fusion cellular assays, making it 17 to over 250-fold more potent than crizotinib. Table 1. Crizotinib and lorlatinib potency against ROS12122
Ki
HCC781 Cell pROS1 IC50 (nM)
BaF32 pROS1 (nM)
Cell IC50
Compound
ROS1 (nM)
Crizotinib
0.6 ± 0.2
51 ± 23
3.9 ± 0.92
Lorlatinib
