the same host. (a) Experimental design. In order to compare the reconstituting ability of p18 -/-

Size: px

Start display at page:

Download "the same host. (a) Experimental design. In order to compare the reconstituting ability of p18 -/-"

Gregory Watts
5 years ago
Views:

1 1

2 Supplementary Figure 1 Direct comparison between p18 -/- and p27 -/- hematopoietic cells in the same host. (a) Experimental design. In order to compare the reconstituting ability of p18 -/- and p27 -/- HSCs, we mixed BMNCs from p18 and p27 knockout mice at a ratio of 1 to 1 ( each), and then injected the cells into lethally irradiated B6.2 hosts. (b) cbmt results. The y-axis shows the ratio of p18 -/- versus p27 -/- cells. p18 -/- HSCs outcompeted p27 -/- HSCs by more than 4 times at 1 year post-bmt (n=10) (p>0.05). (c) Data from one representative recipient of cbmt, as shown in figure S1a. The values in the corner show the ratio of p18 -/- cells versus p27 -/- cells. As shown in the upper panel, p18 -/- dominated in blood, B cell, and myeloid lineages, while p27 -/- dominated in the T cell lineage. The lower panel shows the contribution of both p18 -/- and p27 -/- to different stages of hematopoiesis. 2

3 3

4 4

5 Supplementary Figure 2 Proliferative rates of HSCs and HPCs. (a) Age-matched p18 -/- and p18 +/+ mice drank water containing 1 mg/ml BrdU for 13 weeks. BrdU incorporation (%) in each population of p18 -/- and p18 +/+ mice at different time points (left). Linear regression for the LKS population was analyzed based on the equation: Ln (% of Brdu negative cells) = a + b (Time). The average turnover time is determined when the negative fraction declines to 37% (right). (b) BrdU incorporation in vivo after BMT. BMNCs ( ) from p18 -/- and p18 +/+ were transplanted into lethally irradiated mice, respectively. The mice received 100 µg/g of BrdU via I.P. injection and were sacrificed 16 hours afterward. Donor BMNCs were assayed for BrdU incorporation in different hematopoietic cell subsets using flow cytometry at different time 5

6 points, as indicated. No statistically significant difference between p18 +/+ and p18 -/- was observed at any time point (n=3-5/each). (c-f) Proliferative kinetics of single HSCs in vitro. Single CD34 - KLS cells were sorted into Terasaki plates with serum free medium containing 50 ng/ml SCF, 25 ng/ml FL, and 10 ng/ml TPO. A total of 147 cells from p18 +/+ mice and 168 cells from p18 -/- mice were monitored. Cells in each well were counted daily for 3 days. (c) Frequencies of divided cells ( 2 cells/well); (d) Frequencies of undivided cells (0 o ) and cells that divided once (1 o ) or more (2 o ) at day 3. (e) Distribution of total cell numbers per well at day 3. (f) Overall expansion of the original single cells at day 3. (g) the total number of homed cells (CD45.2) in the BM 17 hours after lin - Sca1 + c-kit + (LSK) cells were injected into the recipients.(h) Flow cytometry analysis of the average percentages of G0, G1, and S/G2/M fractions in CD LKS cells. 6

7 7

8 Supplementary Figure 3 (a) 3D structural model of the p18 protein and docking poses of all tested compounds at the p18 surface binding cavity. (b) Overview of the 3D structural model of 8

9 the p18 protein and docking poses of P18IN011. Purple dashed lines highlight the hydrogen binding. Yellow and blue dashed lines indicate the hydrophobic interaction with F37 and the charge interaction with R39, respectively. (c) The limit dilution analysis of groups treated with P18IN03A or P18IN005. (d) CDK6 activity assay in vitro. Recombinant Rb fragments were used as substrates for the activated complexes of CDK6/ CyclinD1 32. P-labeled GST-Rb was quantitatively analyzed by a scintillation counter and normalized with the mean of the CDK6-Rb group. Supplementary Figure 4 Frequency of CD34 - LKS cells measured by limit dilution assay (LDA). 9

10 Supplementary Figure 5 General chemistry syntheses route of P18IN011 and its analogs. 10

11 Supplementary Table 1 Function of HSCs (CD34-LKS) in the Secondary Recipients (6 months post-cbmt) Genotype Input Output Progeny/HSC (HSCs/mouse) (BMNCs/mouse) p18 -/- 2, ,000 p27 -/ ,600 WT ,000 11

12 Supplementary Note p18 inhibitors discovered. To overcome the intrinsic limitation and low throughput of the current bioassays for screening small molecules and testing HSC self-renewal, we have developed alternative technologies, which have enabled us to discover the first p18 inhibitor using our established target-focused computational chemical genomics screening and in vitro and ex vivo validation approaches. First, we have modeled the 3D protein structure of p18 and its binding surface cavity using our reported protocol 1, 2 based on X-ray crystallographic data 3, 4, 5, 6. The X-ray data show that p18 protein, which consists of five ankyrin repeats, binds next to the ATP binding site of the CDK6 catalytic cleft (residues ), and interacts with both the N-lobe (residues 9-104, rich in β-sheet) and C-lobe (residues, , predominantly α-helix) of the CDK6 kinase. According to structural biology data 7, p18 binding to CDK6 may lead to the dissociation of cyclin D, which binds at the site opposite the CDK6 catalytic cleft (PASTAIRE helix residues: 55-66). In addition, the data show that when the CDK6 is bound to p18, its N- and C-lobes are rotated 13Å away from each other, resulting in the misalignment of ATP binding residues. As a result, the N-lobe PASTAIRE helix domain of CDK6, which contains an invariant active site residue (Glu61), is displaced by 64.5 Å away from the active site, and is rotated by 16 Å. In addition, p18 displaces the N-lobe relative to the C lobe of CDK6, causing the hydrophobic residues (Ile19, 12

13 Val27, Ala41, Leu152) that sandwich the adenine ring of ATP to move by up to 4.5 Å. p18 inhibition of CDK6 also distorts the edge of the CDK6 active site via Phe82, affecting H-bonding interactions with the edge of the ATP ring. Taken together, these results cause us to speculate that binding of p18 to CDK6 disrupts the ATP binding site, as well as the cyclin D binding site in CDK6, thus blocking the activation effects of the CDK6/cyclin complex on the HSC cell cycle. Such important phenomena led us to the hypotheses that identifying a p18 protein blocker can impede p18 binding to CDK6, distort the CDK6 cell cycle, and stimulate the activation effect of the kinase complex on HSC cell self-renewal. Having defined the binding cavity at p18 above, then we conducted 3D docking virtual screening using our reported 3D virtual screening approach 1, 8 and compound library 9. The top 15% of docking score ranked compounds were subjected to manual inspection according to the conserved hydrogen bond with Arg39 or Asp76 of p18. Among them, the lead compounds P18IN003 and P18IN011 have a total score (equivalent logk d ) of 6.80 and 7.38, respectively, which are ranked in the top 5% of the in silico screened compounds. Subsequently, 12 compounds were obtained via MTA or commercially, and biologically tested in vitro for their ability to promote HSC self-renewal, among which 2 compounds (P18IN003 and 13

14 P18IN011) showed medium to great increased activity. The compound P18IN011 (MW , clogp 2.3) was identified as one of the virtual hits by the Surflex-dock database search. The docking pose shows that the hit compound P18IN011 has extensive H-bonding interactions with the key residues, including its amide NH group with D76 (1.90 Å), C=O with Q43 (3.16 Å) and D67 (3.20 Å), sulfonate O=S=O with and T69 (2.71 Å) and its charge interaction with R39 (charge interaction, 3.12 Å) of the p18 protein (Fig. 3c). In addition, hydrophobic interactions were also detected for its isoxazobenzine ring with F37 and V44 of p18 and also the acetamidobenzene ring surrounded by hydrophobic residues F71, A72 and V73 of p18 in a range of 3.3 ~ 3.7 Å. It is known that the residues F37, R39, Q43, V44, D67 and D76 of p18 are highly conserved residues located at the p18/cdk6 interface and they that play important roles in the H-bonding or hydrophobic interactions with CDK6. The identified additional binding residues T69, F71, A72 and V73, are exclusively for p18 based on homology modeling in comparison with other CKI proteins. Thus, by blocking these key residues, a compound is predicted to disrupt the interaction of p18 with CDK6, which has been further confirmed by the biological experiments. Finally, we carried out the CAFC assay with limiting dilution using our reported protocol 10, 11 to functionally validate the effects of the virtual p18 hits above. The confirmatory experiments with 14

15 dose response curves show that compounds P18IN011 and P18IN03 can significantly increase the frequency of HSCs (ED 50, 3.07 and 5.70 µm), suggesting a potential enhancement of HSC self-renewal. References: 1. Chen JZ, Wang J, Xie XQ. GPCR structure-based virtual screening approach for CB2 antagonist search. J Chem Inf Model 47, (2007). 2. Xie* XQ, Chen JZ, Billings EM. 3D structural model of the G-protein-coupled cannabinoid CB2 receptor. Proteins: Structure,Function,and Genetics 53, (2003). 3. Brotherton DH, et al. Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19ink4d. Nature (London) 395, (1998). 4. Jeffrey PD, Tong L, Pavletich NP. Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes & Development 14, (2000). 5. Padmanabhan B, Adachi N, Kataoka K, Horikoshi M. Crystal Structure of the Homolog of the Oncoprotein Gankyrin, an Interactor of Rb and CDK4/6. J Biol Chem 279, (2004). 6. Venkataramani RN, MacLachlan TK, Chai X, El-Deiry WS, Marmorstein R. Structure-based Design of p18ink4c Proteins with Increased Thermodynamic Stability and Cell Cycle Inhibitory Activity. J Biol Chem 277, (2002). 7. Jeffrey PD, Tong L, Pavletich NP. Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors. Genes Dev FIELD Full Journal Title:Genes & Development 14, (2000). 15

16 8. Chen J-Z, Han X-W, Liu Q, Makriyannis A, Wang J, Xie* X-Q. 3D-QSAR Studies of Arylpyrazole Antagonists of Cannabinoid Receptor Subtypes CB1 and CB2. A Combined NMR and CoMFA Approach. Journal of medicinal chemistry 49, (2006). 9. Xie* XQ, Chen JZ. Data mining a small molecule drug screening representative subset from NIH PubChem. J Chem Inf Model 48, (2008). 10. Yu H, Yuan Y, Shen H, Cheng T. Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners. Blood 107, (2006). 11. Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18ink4c. Nat Cell Biol 6, (2004). 16

Supplemental Information Inventory

Supplemental Information Inventory Cell Stem Cell, Volume 6 Supplemental Information Distinct Hematopoietic Stem Cell Subtypes Are Differentially Regulated by TGF-β1 Grant A. Challen, Nathan C. Boles, Stuart M. Chambers, and Margaret A.