Understanding the Species Selectivity of Myeloid cell leukemia-1 (Mcl

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Understanding the Species Selectivity of Myeloid cell leukemia-1 (Mcl-1) Inhibitors Bin Zhao, Allison L. Arnold, Marcelle A. Coronel, Joyce H. Lee, Taekyu Lee, Edward T Olejniczak, and Stephen W. Fesik Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00626 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Biochemistry

Understanding the Species Selectivity of Myeloid cell leukemia-1 (Mcl-1) Inhibitors

Bin Zhao, Allison L. Arnold+, Marcelle A. Coronel+, Joyce H. Lee, Taekyu Lee, Edward T. Olejniczak, and Stephen W. Fesik*

Department of Biochemistry, Vanderbilt University School of Medicine, 2215 Garland Avenue, 607 Light Hall, Nashville, Tennessee 37232-0146, USA + Contributed equally to work. KEYWORDS: apoptosis; cancer; Mcl-1; inhibitor; drug discovery;

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Abstract In order to test for on target toxicity of a new chemical entity, it is important to have comparable binding affinity of the compound in the target proteins from humans and the test species. To evaluate our Mcl-1 inhibitors, we tested them against rodent Mcl-1 and found a significant loss in binding affinity when compared to human Mcl-1. To understand the affinity loss, we used sequence alignments and structures of human Mcl-1/inhibitor complexes to identify the important differences in the amino acid sequences. One difference is human L246 (F226 in rat, F227 in mouse) in the ligand binding pocket. Mutating rat F226 to a Leu recovers affinity, but the mouse F227L mutant still has lower ligand affinity than human Mcl-1. Another change in mouse, F267, located ~12 Å from the ligand pocket, when mutated to the human/rat cysteine, F267C, improved affinity and combined with F227L resulted in a mutant mouse protein with similar binding affinity as human Mcl-1. To help understand the structural components of the affinity loss, we obtained an X-ray structure of a mouse Mcl-1/ inhibitor complex and identified how the residue changes reduced compound complementarity. Finally, we tested the Mcl-1 of other preclinical animal models (canine, monkey, rabbit and ferret) that are identical to human for these two residues and found that their Mcl-1 bound our compounds with affinities comparable to human Mcl-1. These results have implications in understanding ligand selectivity for similar proteins and for the interpretation of preclinical toxicology studies with Mcl-1 inhibitors.


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Biochemistry

Introduction Apoptosis, or the process of programmed cell death, is an essential and normal process of multicellular organisms. It is usually tightly controlled but in cancer, abnormal regulation can contribute to the phenotype of the disease 1, 2. The apoptosis pathway is initiated by various extracellular and intracellular stresses, including growth factor deprivation, DNA damage, oncogene induction, and cytotoxic drugs3. Oligomerization of the pro-apoptotic proteins Bax and Bak, permeabilize the mitochondrial outer membrane and release apoptogenic factors into the cytoplasm such as cytochrome c which results in the activation of a caspase cascade. This process is regulated by both pro-apoptotic (Bax, Bak, Bad, Bid, Bim, Bmf, NOXA, PUMA) and anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w, Bcl2-A1, Mcl-1) members of the Bcl-2 family of proteins that respond to the various extracellular and intracellular stresses4, 5. An important anti-apoptotic member of the Bcl-2 family6 is Myeloid cell leukemia-1 (Mcl-1). Mcl-1 is one of the most frequently amplified anti-apoptotic genes in human cancers7, 8 and is associated with a number of cancers. Mcl-1 overexpression is also implicated as a resistance factor for multiple therapies9 including the Bcl-2 inhibitor venetoclax10.

Compounds which bind to Mcl-1 and inhibit its activity could increase

tumor cell death in Mcl-1-dependent cancers and increase sensitivity to many standard chemotherapeutics2, 11. Recently, we and others have reported on compounds that bind in the binding groove of human Mcl-1 and inhibit its antiapoptotic activity in human cancer cell lines, leading to apoptosis12-18. To evaluate our compounds in vivo, we used mouse xenograph models of human cancers. An important consideration in the in vivo studies is the tolerability of the compounds in the host animal. To help us interpret the tolerability studies, we wanted to know the affinity of our Mcl-1 inhibitors to the endogenous mouse protein to get an indication of the contribution of on target toxicity in the animal tolerability studies. However, when we tested our compounds against mouse and rat Mcl-1, we were surprised to find a significant loss in binding affinity for our compounds. The fold loss in affinity when compared to human Mcl-1, was ~ 200 fold for mouse Mcl-1 and ~30 fold for rat Mcl-1.


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How ligands achieve specificity for very similar homologous proteins is a question of practical interest. For our Mcl-1 drug discovery efforts, understanding this species selective binding activity is important because of it implications for how to interpret toxicity studies in different animal species. Ligand selectivity for very similar proteins is also a topic of great interest in drug optimization where it is often desired to gain specificity on homologous proteins to avoid side effects from inhibiting unwanted proteins19. Here we describe our studies to understand the factors that contribute to the rodent species dependent ligand binding specificity. We first used mutagenesis and structural studies to understand the residue differences responsible for the dramatic decrease in inhibitor binding to rodent Mcl-1. Next, to determine the binding specificity for Mcl-1 derived from other common preclinical animal species, we cloned, expressed, and purified Mcl-1 from these species and tested them for binding to our Mcl-1 inhibitors. From these studies, we were able to identify the residues important for the loss in affinity in rodent, and we identified Mcl-1 proteins from other species that have similar binding affinity of our compounds compared to human Mcl-1. Materials and Methods Construct Design of BH3 Binding Domain of Mcl-1. The Mcl-1 PEST domain (1-171) 20 is not considered part of the BH3 binding domain and was excluded from all of the constructs. The structure of the Mcl-1 BH3 binding domain (172-321) used in this study is compared to the BH3 binding domain of Bcl-xl in Supplementary Material Figure S1. From the structure alignment, it can be seen that Mcl-1 (172-321) has the same fold as the Bcl-xl BH3 binding domain but does not have the long unstructured loop between helix 1 and 2 found in both Bcl-xl and Bcl-2. The proteins also bind the Bim BH3 peptide in analogous binding grooves in the protein. In Supplementary Figure S2, we show a sequence alignment of Mcl-1(172-321) and the Bcl-xl BH3 binding domain after excluding the PEST domain of Mcl-1 and the unstructured loop of Bcl-xl. In the alignment, we have labeled the BH-domains of Bcl-xl


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Biochemistry

above the sequence. All of the Mcl-1 BH3 binding domain constructs from the different species were based on their alignment with human Mcl-1 (172-321). Protein expression and Purification for Assays and X-ray Structures Protein preparation was described previously13, 21 and modified slightly for the Mcl-1 from different species. Briefly, the Mcl-1 constructs were generated using gene synthesis (Genescript) with codons optimized for expression in E. coli. Genes were subcloned into an expression vector (pET28A) expressed in Escherichia coli BL21 Gold (DE3) (Stratagene) and purified through nickel-column and size-exclusion chromatography sequentially. The proteins used in the assay had the general sequence HisMBP-ENLYFQG-Mcl-1(BH3) where the species BH3 binding domain is given in Figure 1. The mouse Mcl-1 for crystallization studies had a different construct with a GlySer linker between MBP and mouse Mcl1 to give HisMBP-GS-mMcl-1(BH3). The linker was based on a human MBP fusion construct described earlier21. Binding Assays A fluorescein isothiocyanate (FITC)-labeled BH3 peptide derived from Bak (FITCBak; FITC-AHx-GQVGRQLAIIGDDINR-NH2) was purchased (Genscript). Measurements used 384-well, white, flat-bottom plates (Optiplate). Signal (Delta F) was measured on a Biotek Cytation 3 equipped with a filter cube containing an Ex 340/30 nM Em 620/10 filter and an Ex 340/30 Em 520 filter. FITC-Bak Peptide Binding to Mcl-1-MBP Fusion Proteins 2 uM FITC-Bak peptide, serially diluted 1:2 in a 12 point curve was incubated with 1 nM Mcl-1-MBP fusion protein and 1 nM Terbium labeled anti-MBP labeled antibody, in 4.52 mM Monobasic potassium phosphate, 15.48 mM dibasic potassium phosphate, 1 mM sodium EDTA, 0.05% Pluronic F-68, 50 mM NaCl, 1 mM DTT, pH 7.5. FITC-Bak was diluted in buffer (12-point, 1:2 serial dilutions) and added to assay plate with protein and antibody, incubated for 3 hours and signal (Delta F) was measured on the Biotek Cytation 3. IC50 values were calculated using XLFit (IDBS) and converted into a binding ACS Paragon Plus Environment

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dissociation constant. Two or more repeats were obtained, and average values are reported. A table of binding constants of the FITC-BAK peptide probe to all of the proteins in this study is given in Supplemental Table S2. Ligand Binding Competition Assays 300 nM FITC-Bak peptide, 1 nM Mcl-1-MBP fusion protein, and 1 nM anti MBP-terbium (Cisbio, Bedford, MA, USA), were added to a buffer containing 0.5 mM monobasic potassium phosphate, 15.5 mM dibasic potassium phosphate, 1 mM sodium EDTA, 50 mM sodium chloride, 1 mM DTT and 0.05% Pluronic F-68 (Sigma-Aldrich, St. Louis, MO, USA) adjusted to pH 7.5. Compounds were diluted in DMSO in a 10-point, semilog serial dilution scheme, and incubated with the protein peptide mixture in 384 well plates for 3 hours. The change in TR-FRET signal (Delta F) was measured on the Biotek Cytation 3 and used to calculate an IC50 (inhibitor concentration at which 50% of bound probe is displaced) by fitting the Delta F values using XLFit (IDBS) to a four parameter dose-response (variable slope) equation. This was converted into a binding dissociation constant22 to give a Ki. Two or more repeats were obtained, and average Ki values are reported. Protein crystallization, Data collection, and Structure refinement Structural studies were performed as previously described13. Briefly, mouse Mcl-1 protein (10 mg/mL) was mixed with a 10 fold excess of ligand in solution (15-20% PEG 6000, 0.1 M TRIS-Hcl pH 8.5) by sitting drop followed by flash freezing after cryoprotection using 20% ethylene glycol. Data were collected at Life Sciences Collaborative Access Team (LS-CAT) 21-IDG beamline, Advanced Photon Source (APS), Argonne National Laboratory. Indexing, integration, and scaling were performed using HKL2000 (HKL Research)23, phasing was accomplished by molecular replacement with Phaser (CCP4)24, 25 using the structure (PDB: 4WMU) as a model, and refinement used Phenix26. Structural statistics are given in the Supplementary Material. Figures were prepared with PyMOL (Schroedinger, LLC: New York, 2010)27.


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Biochemistry

Results and Discussion Binding Affinity for Rodent Mcl-1 When we tested our human Mcl-1 inhibitors in an in vitro binding assay against rodent Mcl-1, we found a dramatic drop in binding affinity (Table 1). The loss in affinity for compound 1 was over 200 fold for mouse and 30 fold for rat when compared to human Mcl-1. When we tested the FITC-BAK BH3 (Bcl-2 homology domain 3) peptide, that mimics the binding of Mcl-1’s natural binding partner, we also observed a greater than four fold affinity loss for mouse when compared to human Mcl-1 (Supplementary Material Table S2).


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Table 1. Ligand Binding to human/rodent Mcl-1 BH3 binding domains (human 172321)

1

2

Ki(nM) N N

3

Ki(nM)

HO

Cl

Ki(nM)

N N

O

Proteins

abs

O

N

O N

O N

N

Ki(nM)

N N

Cl

N

4

N

N

Cl

N

OH

N

OH O

O

O

N O

O

HO

O

Cl

Cl

O

O

Cl

Human Mcl-1

0.032+/-0.01

0.36+/-0.02

0.34+/-0.02

66. +/-7.0

WT Mouse

7.37+/-0.6

52+/-9.

52+/-8

>75000

Mutant Mouse F227L Mutant Mouse F267C

0.15+/-0.4

5.4+/-0.5

5.2+/-0.1

341 +/-30

4.9+/-0.9

35.2+/-0.4

36+/-4

15000+/10000

Double Mutant Mouse F267C F227L

0.24 +/-0.01

1.4 +/- 0.2

2.0 +/- 0.2

n.d.

Rat

0.95+/-0.2

9.6+/-0.7

12.3 +/- 0.5

>75000.

Rat F227L

0.06+/-0.01

0.3+/-0.02

0.45+/-0.1

112+/-20

n.d.=not determined


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Biochemistry

This species dependent loss in binding affinity for our Mcl-1 inhibitors was not expected based on sequence alignments. As shown in Figure 1, the sequences of the human and the two rodent species Mcl-1 binding domains (human Mcl-1, 177-296 ) are very similar with only a small number of residues that differed compared to human (color coded blue in the alignment with hMcl-1). The sequences are almost identical when looking at residues in the binding groove that contact the BH3 peptides in human Mcl-1/peptide structures (e.g. Bak,Bim) (color coded red). From the sequence comparison alone, it was difficult to determine which residue differences were responsible for the loss in affinity and to explain why mouse Mcl-1 binds ligands more than seven fold weaker than rat Mcl-1.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Multispecies alignment of Mcl-1 BH3 binding domains. Residues near the Bim BH3 peptide in human Mcl-1/complexes are colored red. Blue colored residues are species differences. Boxes are placed around residue differences discussed in text.


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Biochemistry

We next used the compound SAR (structure activity relationship) and structural data on how the compounds bind to human Mcl-113, 28 to help identify the sequence differences in rodent and human Mcl-1 that could be responsible for the changes in affinity. From the SAR, we hypothesized that the loss in affinity to rodent Mcl-1 is dominated by the 3 indole substitution. Compounds with the di-methyl chloro phenyl substituent (1-3) decrease binding ~200 fold (Table 1) while compound 4 with a napthal as the indole-3 position substituent, has a thousand fold decrease in binding to mouse when compared to human Mcl-1. The Bak peptide which contains a smaller Leucine substituent that is located in the P2 pocket,29 has a much smaller fold change in binding affinity. When we compared the binding mode of compound 2, (cyan)(PDB ID: 5IF4 )13, and the Bim BH3 peptide (salmon)(PDB ID: 2PQK ) in Figure 2, the compound binds in the peptide binding groove with the di-methyl chloro phenyl substituent penetrating deep into the P2 pocket. The binding of compound 2 causes multiple changes in the P2 pocket of human Mcl-1 including a bend in helix 4 (Figure 2B) which results in the top of the helix being closer to the 3 indole substitution of the ligand. Compound binding also results in an extra turn in the C-terminus of helix 6 when compared to the Bim (pdb id 2PQK) structure (Figure 2A). The most important difference suggested by the structural data for 2 is the L246 to F227/F226 mouse/rat residue change deep in the P2 pocket. From the structure, it can be seen that the di-methyl-Cl Phenyl group reaches down to just above L246 on helix 4 (Figure 2B). This sequence difference deep in the P2 pocket could thus contribute to most of the loss in binding affinity in mouse/rat when compared to human Mcl-1.


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Figure 2. Comparison of X-Ray Structural data for the Mcl-1 inhibitor, 2, and the Bim peptide bound to human Mcl-1 (hMcl-1). (A) Superposition of 2 (green) (PDB ID: 5IF4) and Bim peptide structure (PDB ID: 2PQK) when bound to hMcl-1. The hydrophobic pockets P2-P4 are labeled and important peptide residues are rendered with magenta sticks. (B) Compound 2 (cyan) bound to hMcl-1. Red arrows highlight bend in helix 4, the circled and rendered residues, L246 and C286, are Phenylalanine in mouse.


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Biochemistry

Mutational Studies on Mouse and Rat Mcl-1 To test the effect of the F227 residue difference in mouse and the equivalent residue F226 in rat, we mutated them to a Leucine, to match the human Mcl-1 protein. When ligand binding was retested, this single mutation recovers much of the ligand binding affinity for rat Mcl-1. However, the mouse Mcl-1 F227L mutant still has about a ten fold lower binding affinity than human (Table 1). To explain this, we looked for other differences in the rodent sequences present in mouse but not in rat/human. As can be seen in the sequence alignment (Figure 1), mouse Mcl-1 contains another residue (F267) that is different between human and rat located in the C-terminus of helix-6. In human, this residue is a cysteine (C286) and it is also a cysteine (C266) in rat. Although this residue is far from the ligand binding site, it becomes part of the extra turn in the C-terminus of helix 6 when compound is bound to human Mcl-1 (Figure 2A). This ligand induced conformational change suggested that a residue change at this position in mouse may also influence compound affinity even though C286/F267 is more than 12 Å away from the ligand. The effect of the C286/F267 change in mouse was tested by the mutation F267C and when the protein was retested, we found this mutation improved ligand binding by 1.5 fold compared to wild type (wt) mouse. When the two mutations, F227L and F267C, were combined in mouse Mcl-1, the ligand binding affinity of 1 is improved about seventy fold and almost matched the affinity of the human protein (Table 1). These mutational studies indicated that F227L and F267C have the biggest effect on mouse Mcl-1 ligand affinity but additional sequence differences in mouse Mcl-1 may still contribute a small amount to the remaining difference in ligand binding. Crystal Structure of Mouse Mcl-1 in Complex with Compound 2 To understand the structural contribution of these residue differences, we performed Xray structural studies on ligand complexes of wt-mouse Mcl-1. Previous X-ray structures of mouse Mcl-1 reported in the literature are for a mouse/human chimera which contains the humanized F227/L246 and F267/C286 mutations30. To obtain a wt-mouse structure we tested multiple ligands and protein constructs and only compound 2 in complex with a mouse Mcl-1– MBP fusion construct21 was successfully crystallized.


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In the X-ray structure, there are eight copies of mouse Mcl-1/ligand 2 molecules in the asymmetric unit cell. Each copy has different crystal contacts but overall the conformations of the protein/ligand copies are similar. Copy D was identified as having the fewest crystal contacts and was used for figures and the discussion. A comparison of the structures of ligand 2 in human Mcl-1 and mouse Mcl-1 is shown in Figure 3A.


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Biochemistry

Figure 3. Ligand binding of compound 2 in human and mouse Mcl-1. (A) Superposition of the ligand complex of 2 in mouse Mcl-1 and human Mcl-1. Helix 4 in mouse magenta, in human green. (B ) Residues V249,V253 in human (green) and equivalent residues in mouse (magenta) are circled.


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Because of the observed loss in binding affinity of the ligand to mouse Mcl-1, it was surprising that the ligand in both the mouse and human protein/ligand complexes superimpose almost exactly. In particular, the 3–indole di-methyl chloro phenyl substitution goes equally as deep into the P2 pocket of both protein complexes. The side chain of L246/F227 also has the same side chain rotamer with the residue sitting just below the dimethyl-Cl phenyl group of the ligand (Figure 3A). These observations suggest that the affinity loss is not due to changes in conformation of the ligand upon binding. However, as shown in Figure 3A, the methyl groups of L246 in human Mcl-1 may have a more favorable binding interaction than the Phe in mouse/rat. Significant changes in structure are observed for the protein. In the mouse structure, the whole helix-4 shifts about 1.5 angstrom out of the pocket when compared to the helix in the human Mcl-1 ligand structure. The shift may be caused by the bigger side chain of the Phe at the L246 position in rodent. In the human structure, replacing the Leu with a Phe would cause a bump with residue I237 on helix-3 unless helix-4 moved away, which is what we observe in the mouse structure. Although both mouse and human ligand binding cause helix- 4 (green helix human, magenta helix mouse) to bend towards the ligand, the angle of the bend is different. The bend in helix-4 also starts near the human/mouse sequence difference L246/F227 which sits between helix-4 and helix-3. One consequence of the movement of helix-4 is an increase in the distance of contacting residues like V249 and V253 to the ligand in the mouse Mcl-1 complex (Figure 3B) which would contribute to the decrease in favorable binding energy for rodent Mcl-1. The comparison of the mouse/human ligand structure also helped provide an explanation for the structural role of the distal residue difference, C286/F267. As seen in Figure 4A, ligand binding in both human and mouse Mcl-1 cause the N-terminus of helix-4 and the C-terminus of helix-6 to gain an extra turn when compared to the Bim BH3 peptide/human Mcl-1 complex. C286 becomes part of the extra turn of helix-6 and points towards the end of helix-4 and the loop connecting helix-4 to helix-3. In the mouse Mcl-1 ligand complex, we observed a displacement of the loop (circled in Figure 4B) and the end of helix-4 when compared to human Mcl-1. The larger side chain, F267 in mouse, may be


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Biochemistry

responsible for this difference. This structural change likely contributes to the overall displacement of helix-4 (Figure 3B ) away from the ligand in mouse when compared to human Mcl-1. From a detailed analysis of the two structures it can be seen how the L246/F227 and the distal residue change (C286/F267) trigger a cascade of conformational changes that result in reduced compound complementarity. These structural differences in the proteins help explain the observed affinity differences for ligand binding between mouse and human Mcl-1.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Ligand binding of compound 2 in human and mouse Mcl-1. (A) Complex in mouse compared to human Mcl-1 Bim peptide structure. Residue F267 of mouse and the loop connecting helix 3-4 are circled. (B) Superposition of Complex 2 in mouse and human Mcl-1. F267 of mouse and adjacent loop connecting helix 3-4 are circled. Human helix 4 green and mouse magenta.


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Biochemistry

Testing Compounds to the MCL-1 of other Animal Species In addition to rodent, several other animal species have been used for toxicological studies. In Figure 1, we compare the sequence alignment of canine, monkey, rabbit and ferret to the human and rodent species Mcl-1 BH3 (Bcl-2 homology 3) binding domains. All of these non-rodent orthologs are very similar to human Mcl-1 but they still have a few conservative residue differences. We cloned and expressed the Mcl-1 proteins from four species (ferret, rabbit, rhesus monkey, and canine) and then tested them in our in vitro binding assay. As shown in Table 2, we found that they bound ligands almost as tightly as human Mcl-1. Looking at the sequence alignment (Figure 1), it can be seen that all of these non-rodent species contain identical residues to those in human at L246 and C286, which we found caused the affinity loss in mouse Mcl-1.


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Table 2. Ligand Binding to human and other species Mcl-1 BH3 binding domain (corresponding to human residues 172-321 )

Proteins

1

2

3

4

Ki(nM)

Ki(nM)

Ki(nM)

Ki(nM)

N N

HO

N N

O

Cl

O

N N

Cl

N

abs

N

N O

N

N

O N

N O

Cl

N

OH

N

OH O

O N

O

Cl

O

HO

O

O

Cl

O

Cl

Human Mcl-1

0.032+/-0.01

0.36+/-0.02

0.34+/-0.02

66 +/-7

Ferret

0.035+/-0.006

0.55+/-0.03

0.37+/-0.02

87+/-20

Rabbit

0.041+/-0.006

0.70+/-0.03

0.41+/-0.03

67+/-9

Rhesus Monkey

0.048+/-0.003

0.43+/-0.02

0.27+/-0.04

59 +/-10

Canine

0.050+/-0.002

0.73+/-0.15

0.45+/-0.1

69+/-4


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Biochemistry

Conclusion Using sequence alignments, mutations and structures of Mcl-1/inhibitor complexes, we identified two important residue differences that cause a substantial loss in ligand affinity for mouse Mcl-1. One of the residues is more than 12 Å away from the ligand yet it still influences the ligand binding pocket. We also looked at the sequences of other preclinical animal models: canine, monkey, rabbit and ferret, where we found that unlike rodent they were identical to human at these two residues and when our inhibitors were tested against their Mcl-1 they had similar ligand affinity as human. The loss of ligand affinity in rodents that we describe here has also been observed for other Mcl-1 inhibitors. Recently, data for an Mcl-1 inhibitor, S63685 has been published, and like our inhibitors it also contains a substituent that points into the P2 pocket. The substituent does not go as deeply into the P2 pocket as our ligands 12 but it still has a six fold loss in affinity to mouse Mcl-1 when compared to human12. The results obtained here for protein species orthologs also has implications for how ligands can gain specificity between close human protein homologs. Many examples exist of drugs that achieve better than a ten fold target selectivity for homologous proteins despite very high homology in the binding pocket15, 19. In many cases, differences in residues in the second shell around the binding pocket are implicated in the affinity differences. These results may also have implications for Mcl-1 inhibitors in rat/mouse animal efficacy, toxicology, and preclinical studies. As shown here, inhibitors designed to target human Mcl-1 can have much less effect on the rat/mouse proteins making them poor choices for evaluating the on target toxicity of these compounds. However, based on our study of inhibitor binding to Mcl-1 from other commonly used preclinical animal models, these other preclinical species could serve as better models than rodent to evaluate on target compound toxicity.


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ASSOCIATED CONTENT PDB ID codes: 6DM8 Supporting Information. The Supporting Information associated with this manuscript is available free of charge on the ACS Publications website. File includes: Table S1. X-Ray data collection and refinement statistics, Table S2. Table of Kd of human FITC-Bak BH3 Peptide to proteins used in this study, Figure S1. Structural comparison of the Mcl-1 BH3 domain (172-321) used in this study and the Bcl-xl BH3 binding domain, Figure S2. Sequence Alignment of the Mcl-1 BH3 domain (172-321) used in this study and Bcl-xl BH3 binding domain.

AUTHOR INFORMATION Corresponding Author *Phone: +1 (615) 322 6303. Fax: +1 (615) 875 3236. Email:[email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors thank co-workers at the High-Throughput Screening Core facility of Vanderbilt University, TN, for compound management and Zhiguo Bian, Subrata Shaw Christopher Tarr and Jason Burke for compound preparation and William Payne for protein purification. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Chemical Biology


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Biochemistry

Consortium Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was also supported by a career development award to S.W.F. from a NCI SPORE grant in breast cancer (Grant P50CA098131) to C. L. Arteaga. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. A portion of the experiments described here used the Vanderbilt PacVan biomolecular robotic crystallization facility, which was supported by National Institutes of Health Grant S10 RR026915. ABBREVIATIONS Mcl-1, myeloid cell leukemia 1; Bak, Bcl-2 homologous antagonist killer; FITC, fluorescein isothiocyanate; Bim , Bcl-2 interacting mediator of cell death ; BH3, Bcl-2 Homology 3; SAR ,structure activity relationship.

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Biochemistry

Figure 1. Multispecies alignment of Mcl-1 BH3 binding domains. Residues near the Bim BH3 peptide in human Mcl-1/complexes are colored red. Blue colored residues are species differences. Boxes are placed around residue differences discussed in text. 114x85mm (300 x 300 DPI)


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Figure 2. Comparison of X-Ray Structural data for the Mcl-1 inhibitor, 2, and the Bim peptide bound to human Mcl-1 (hMcl-1). (A) Superposition of 2 (green) (PDB ID: 5IF4) and Bim peptide structure (PDB ID: 2PQK) when bound to hMcl-1. The hydrophobic pockets P2-P4 are labeled and important peptide residues are rendered with magenta sticks. (B) Compound 2 (cyan) bound to hMcl-1. Red arrows highlight bend in helix 4, the circled and rendered residues, L246 and C286, are Phenylalanine in mouse. 101x90mm (300 x 300 DPI)


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Figure 3. Ligand binding of compound 2 in human and mouse Mcl-1. (A) Superposition of the ligand complex of 2 in mouse Mcl-1 and human Mcl-1. Helix 4 in mouse magenta, in human green. (B ) Residues V249,V253 in human (green) and equivalent residues in mouse (magenta) are circled. 101x73mm (300 x 300 DPI)


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Figure 4. Ligand binding of compound 2 in human and mouse Mcl-1. (A) Complex in mouse compared to human Mcl-1 Bim peptide structure. Residue F267 of mouse and the loop connecting helix 3-4 are circled. (B) Superposition of Complex 2 in mouse and human Mcl-1. F267 of mouse and adjacent loop connecting helix 3-4 are circled. Human helix 4 green and mouse magenta. 101x73mm (300 x 300 DPI)


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Biochemistry

Table of Content Graphic 1.75"x1.75" 44x44mm (300 x 300 DPI)


Understanding the Species Selectivity of Myeloid cell leukemia-1 (Mcl

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