Superposition of selected Mcl-1 structures.

<p>(A) Structures of hMcl-1(171–327) (green) and mMcl-1(152–308) (cyan, PDB accession code 1wsx) after superposition of the backbone N, C^α and C’ atoms of the α-helices for minimal rmsd. (B) Ribbon drawing (zoomed into (A)) showing the different binding groove widths of human (green) and mouse (cyan) protein. The distances between the Cα-atoms of residues His 224 in helix α2 (His 205 in mMcl-1) and His 252 (His 233 in mMcl-1) at the C-terminus of helix α3 are highlighted: ∼16 Å in hMcl-1(171–327) and ∼14 Å mMcl-1(152–308) (C) Superposition as in (A) of hMcl-1(171–327) (green) and mMcl-1(152–308) (cyan, 1wsx), and six selected Mcl-1 complex structures (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone-0096521-t002" target="_blank">Table 2</a>): human Mcl-1 complexed with Bim BH3 (magenta, 2nla); chimeric rat-human rMcl-1(171–208)hMcl-1(209–327) complexed with mouse mNoxaB BH3 (yellow, 2rod); mouse mMcl-1(152–308) complexed with mouse NoxaA BH3 (pink, 2roc); mouse mMcl-1(152–308) complexed with mouse Puma BH3 (grey, 2jm6); mouse mMcl-1(152–308) complexed with mouse NoxaB BH3 (purple, 2rod); chimeric rat-human mMcl-1(171–208)hMcl-1(209–327) complexed with human Bim BH3 (orange, 2nl9); chimeric rat-human mMcl-1(171–208)hMcl-1(209–327) complexed with human Bim BH3(L62A, F68A) (light green, 3d7v).The figures were prepared with the programs MOLMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Koradi1" target="_blank">[36]</a> and PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Delano1" target="_blank">[37]</a>.</p

Electrostatic surface potentials.

<p>(<b>A</b>) For human hMcl-1(171–327) in the orientation shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone-0096521-g001" target="_blank">Figure 1</a> (left) and after rotation by 180° about the vertical axis (right). Surface colors (blue for positively charged; red for negatively charged) indicated the electrostatic potential calculated by using PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Delano1" target="_blank">[37]</a> and its default vacuum electrostatics protocol. (<b>B</b>) Same as in (A) but for mouse mMcl-1(152–308).</p

Statistics of hMcl-1(171–327) NMR Structure.

a<p>Related to pairs with non-degenerate chemical shift.</p>b<p>Regular secondary element: α-helical residues 173–191, 204–235, 240–253, 262–280, 284–301, 303–308 and 311–319.</p>c<p>Ordered residues: 172–192,194–198, 204–235, 238–255, 262–321 with dihedral angle order parameters S(φ) and S(ψ) > 0.90. Z-scores were computed relative to corresponding structure quality measures for high resolution X-ray crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Bhattacharya1" target="_blank">[42]</a>.</p

Rmsd values for comparison of the NMR structure of hMcl-1(171–327) with the structures of mouse mMcl-1(152–308) and Mcl-1complexes.^a

a<p>Average pairwise rmsd values (Å) were calculated for backbone heavy atoms N, C^α, and C’ between the 20 conformers of Mcl-1(171–327) and corresponding polypeptide segments in the other structures. The distances dCA (in Å) between the C^α-atoms of residues His 224 in helix α2 (His 205 in mMcl-1) and His 252 (His 233 in mMcl-1) at the C-terminus of helix α3 are provided as a measure for the width of the BH3 binding groove.</p>b<p>Residue numbers are for hMcl-1(171–327); residues 194–202 were excluded since one structure (2nl9^k) does not contain the corresponding residues; residues 172–193 and 203–321 correspond to residues 153–174 and 184–302 in mMcl-1, and residues 209–321 correspond to residues 190–302 in mMcl-1.</p>c<p>Helices α1–α7 in hMcl-1 comprise residues 173–191, 204–235, 240–253, 262–280, 284–301, 303–308 and 311–319; the corresponding residues in mMcl-1 are: 155–172, 185–216, 221–234, 243–261, 265–282, 284–289 and 292–300.</p>d<p>Helices α2–α7 in hMcl-1 and residues 204–208 (numbers in hMcl-1) were excluded</p>e<p>Mouse mMcl-1(152–308), PDB accession code 1wsx (the mean NMR coordinates were used) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Day1" target="_blank">[10]</a>.</p>f<p>Human hMcl-1 complexed with human hBim BH3, 2pqk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>g<p>Chimiric rat-human rMcl-1(171–208)hMcl-1(209–327) complexed with mouse mNoxaB BH3, 2nla <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Czabotar1" target="_blank">[9]</a>.</p>h<p>Mouse mMcl-1 complexed with mouse mNoxaA BH3, 2rod <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Day2" target="_blank">[12]</a>.</p>i<p>Mouse mMcl-1 complexed with mouse mPuma BH3, 2roc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Day2" target="_blank">[12]</a>.</p>j<p>Mouse mMcl-1 complexed with mouse mNoxaB BH3, 2jm6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Czabotar1" target="_blank">[9]</a>.</p>k<p>Chimiric rat-human Mcl-1 complexed with human hBim BH3, 2nl9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Czabotar1" target="_blank">[9]</a>.</p>l<p>Chimiric rat-human Mcl-1 complexed with human hBim (L62A, F68A), 3d7v <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Lee1" target="_blank">[13]</a>.</p>J<p>Human hMcl1 complexed with human Bid BH3, 2kbw <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Liu1" target="_blank">[15]</a>.</p>k<p>Human hMcl-1 complexed with human Bim BH3 mutant I2dY, 3kj0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>l<p>Human hMcl-1 complexed with human BimL12Y, 3io9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Lee2" target="_blank">[16]</a>.</p>m<p>Human hMcl1 complexed with human Bim BH3 mutant I2dA, 3kj1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>n<p>Human hMcl1 complexed with human Bim BH3 mutant F4aE, 3kj2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Fire1" target="_blank">[11]</a>.</p>o<p>Human hMcl-1 complexed with Mcl1 specific selected peptide B7, 3kz0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Dutta1" target="_blank">[41]</a>.</p>p<p>Human hMcl-1 complexed with human Mcl1 BH3, 3mk8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Stewart1" target="_blank">[17]</a>.</p>q<p>Human hMcl1 complexed with human Bax BH3, 3pk1.</p>r<p>Mouse mMcl-1 complexed with mouse Noxa BH3, 4g35 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Muppidi1" target="_blank">[18]</a>.</p>s<p>Human hMcl-1 complexed with 6-chloro-3-[3-(4-chloro-3,5-dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid, 4hw2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Friberg1" target="_blank">[14]</a>.</p>t<p>Human hMcl-1 complexed with 6-chloro-3-[3-(4-chloro-3,5-dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid, 4hw3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Friberg1" target="_blank">[14]</a>.</p>u<p>Human hMcl-1 complexed with human Mcl1 BH3, 4hw4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Friberg1" target="_blank">[14]</a>.</p

NMR structure of hMcl-1(171–327).

<p>(<b>A</b>) Backbone of the 20 CYANA conformers representing the solution structure of hMcl-1(171–327) after superposition of backbone N, C^α and C’ atoms of the α-helices for minimal rmsd. The three BH sequence motifs are colored in green (BH3), red (BH1) and blue (BH2), respectively. (<b>B</b>) Ribbon drawing of the lowest energy conformer of hMcl-1(171–327). α-helices α1-α7 are labeled and colored differently, and the N- and C-termini are labeled as “<i>N’</i> ” and “<i>C’</i> ”. The figures were generated using the programs MOLMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Koradi1" target="_blank">[36]</a> and PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096521#pone.0096521-Delano1" target="_blank">[37]</a>.</p

Fragment Based Drug Discovery: Practical Implementation Based on ¹⁹F NMR Spectroscopy

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing ¹H–¹⁵N heteronuclear single quantum coherence (HSQC) as well as one-dimensional ¹H and ¹⁹F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of ¹⁹F detection in FBS has been only recently introduced (Vulpetti et al. <i>J. Am. Chem. Soc.</i> <b>2009</b>, <i>131</i> (36), 12949–12959) with the aim of targeting “fluorophilic” sites in proteins. Here, we demonstrate a more general use of ¹⁹F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure–activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability

Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the Na_V1.7 Inhibitory Peptide GpTx‑1

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit Na_V1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with Na_V1.7 and related Na_V subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on Na_V1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a Na_V1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work

Ordering of the N‑Terminus of Human MDM2 by Small Molecule Inhibitors

Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10–16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel β-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors

Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the Na_V1.7 Sodium Channel

Na_V1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on Na_V1.7 (IC₅₀ = 10 nM) and promising selectivity against key Na_V subtypes (20× and 1000× over Na_V1.4 and Na_V1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp²⁹, Lys³¹, and Phe³⁴ near the C-terminus are critical for potent Na_V1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against Na_V1.4. A structure-guided campaign afforded additive improvements in potency and Na_V subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]­GpTx-1 with a Na_V1.7 IC₅₀ value of 1.6 nM and >1000× selectivity against Na_V1.4 and Na_V1.5