Main-chain Nitrogen Research Articles

Inhibition of HIV-1 Protease by a Boronic Acid with High Oxidative Stability.

HIV-1 protease is an important target for pharmaceutical intervention in HIV infection. Extensive structure-based drug design led to darunavir becoming a key chemotherapeutic agent. We replaced the aniline group of darunavir with a benzoxaborolone to form BOL-darunavir. This analogue has the same potency as darunavir as an inhibitor of catalysis by wild-type HIV-1 protease and, unlike darunavir, does not lose potency as an inhibitor of the common D30N variant. Moreover, BOL-darunavir is much more stable to oxidation than is a simple phenylboronic acid analogue of darunavir. X-ray crystallography revealed an extensive network of hydrogen bonds between the enzyme and benzoxaborolone moiety, including a novel direct hydrogen bond from a main-chain nitrogen to the carbonyl oxygen of the benzoxaborolone moiety that displaces a water molecule. These data highlight the utility of benzoxaborolone as a pharmacophore.

Influence of the conformations of αA-crystallin peptides on the isomerization rates of aspartic acid residues

The isomerization rate of aspartic acid (Asp) residue is known to be affected by the three-dimensional structures of peptides and proteins. Although the isomerized Asp residues were experimentally observed, structural features which affect the isomerization cannot be elucidated sufficiently because of protein denaturation and aggregation. In this study, molecular dynamics (MD) simulations were conducted on three αA-crystallin peptides (T6, T10, and T18), each containing a single Asp residue with different isomerization rate (T18 > T6 > T10) to clarify the structural factors of Asp isomerization tendency. For MD trajectories, distances between side-chain carboxyl carbon of Asp and main-chain amide nitrogen of (n + 1) residue (Cγ–N distances), root mean square fluctuations (RMSFs), and polar surface areas for main-chain amide nitrogen of (n + 1) residues (PSAN) were calculated, because these structural features are considered to relate to the formations of cyclic imide intermediates. RMSFs and PSAN are indexes of peptide backbone flexibilities and solvent exposure of the amide nitrogen, respectively. The average Cγ–N distances of T10 was longer than those of the other two peptides. In addition, the peptide containing Asp residue with a higher isomerization rate showed higher flexibility of the peptide backbone around the Asp residue. PSAN for amide nitrogen in T18 were much larger than those of other two peptides. The computational results suggest that Asp-residue isomerization rates are affected by these factors.

Computational studies on nonenzymatic succinimide-formation mechanisms of the aspartic acid residues catalyzed by two water molecules

In the biological proteins, aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. Asp-residue isomerization causes the aggregation and the insolubilization of proteins, and is considered to be involved in various age-related diseases. Although Suc intermediate was considered to be formed by nucleophilic attack of the main-chain amide nitrogen of N-terminal side adjacent residue to the side-chain carboxyl carbon of Asp residue, previous studies have shown that the nucleophilic attack is more likely to proceed via iminol tautomer when the water molecules act as catalysts. However, the full pathway to Suc-intermediate formation has not been investigated, and the experimental analyses for the Asp-residue isomerization mechanism at atomic and molecular levels, such as the analysis of the transition state geometry, are difficult. In the present study, we computationally explored the full pathways for Suc-intermediate formation from Asp residues. The calculations were performed two types of reactant complexes, and all energy minima and TS geometries were optimized using B3LYP density functional methods. As a result, the SI-intermediate formation was divided into three processes, i.e., iminolization, cyclization, and dehydration processes, and the activation energies were calculated to be 26.1 or 28.4 kcal mol−1. These values reproduce the experimental data. The computational results show that abundant water molecules in living organisms are effective catalysts for the Asp-residue isomerization.

Porphyrin–Amino Acid Conjugates

An efficient way for the formation of a direct bond between a porphyrin and amino acid through a main chain nitrogen has been optimized and applied in the synthesis of a library of chromophore-amino acid conjugates with slightly modified and adjustable optical properties. The obtained series of hybrid materials shows an optic response characteristic for meso-nitrogen derivatized porphyrinoids. The oxidation with hypervalent iodine shows a regioselective transformation of an amino acid chain changing the character of the final molecule.

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.

S···O and S···N Sulfur Bonding Interactions in Protein-Ligand Complexes: Empirical Considerations and Scoring Function.

Sulfur bonding interactions between organosulfur compounds and proteins were examined using crystal structures deposited to-date in the PDB. The data was analyzed as a function of sulfur-σ-hole-bonding (i.e., sulfur bonds) to main chain Lewis bases, viz. oxygen and nitrogen atoms of the backbone amide linkages. The analyses also included an examination of sulfur bonding to side chain Lewis bases (O, N, and S) and to the "non-classical" Lewis bases present in electron-rich aromatic amino acids as-well-as to donor-acceptor bond angle distributions. The interactions analyzed included those restricted to the sum of van der Waals radii of the respective atoms or to a distance of 4 Å. The surveyed data revealed that sulfur bonding tendencies (C-S-C bond angles) were impacted not only by steric effects but perhaps also by enthalpic features present in both the donor and acceptor participants. This knowledge is not only of fundamental interest but is also important in terms of materials and drug-design involving moieties incorporating the sulfur atom. Additionally, a new empirical scoring function was developed to address the anisotropy of sulfur in protein-ligand interactions. This newly developed scoring function is incorporated into AutoDock Vina molecular docking program and is valuable for modeling and drug design.

Amino acid residues that affect the basicity of the catalytic glutamate of the hydrolytic aldehyde dehydrogenases

In the catalytic mechanism of hydrolytic aldehyde dehydrogenases (ALDHs) the role of Glu268 (mature human ALDH2 numbering) as a general base is of major relevance. Since Glu268 basicity depends on its protein environment, here we explore its interactions with other amino acid residues in the three different conformations observed in ALDH crystal-structures: “inside”, “intermediate” and “outside”. In all of them Glu268 is in a hydrophobic environment. In the “inside” conformation, the theoretical pKa estimated by PROPKA3 is the result of the effects of hydrogen bonds with the protonated thiol of the catalytic Cys302 and/or the main-chain amide nitrogen of the highly conserved Gly270, and of charge-charge interactions with neighboring side-chains—Lys178, Glu/Asp476, His465 or Glu399 depending on the enzyme. In the “intermediate” conformation Glu268-carboxyl pKa is influenced by interactions with Glu/Asp476, Arg/Lys475, Lys/Arg178, His465 or Arg459, also depending on the enzyme. In the “outside” conformation, the effects on Glu268-carboxyl pKa arise from hydrogen bonds with the side chains of the strictly conserved Thr224 and/or of Lys/Arg178, and from charge-charge interactions with Lys/Arg/Asp178, Glu476, or Arg459. The estimated pKas and interactions of Glu268-carboxyl in the “intermediate” and “outside” conformations are consistent with their previously proposed roles in activating the hydrolytic water and in a proton relay mechanism, respectively. Water channels connecting Glu268 with the bulk water were found in all hydrolytic ALDHs. In the “inside” conformation the theoretical pKas of the Glu268-carboxyl and Cys302-thiol groups suggest that the carboxyl cannot receive the proton from the thiol. We propose that a protonated Cys302 might perform the nucleophilic attack on the aldehyde, which can be facilitated by Glu268 in the “intermediate” conformation. Finally, the conservation of the residues influencing Glu268 basicity between and within ALDH families suggests that these residues, not previously studied, are important for the catalytic mechanism of many ALDH enzymes.

Abstract 4854: Fibulin5 as a novel therapeutic target for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a challenging disease with a five-year survival rate less than 5% due to late diagnosis, aggressive local and metastatic progression and high resistance to chemotherapy. Desmoplasia is thought to be a major contributor to chemoresistance in PDA. However, desmoplasia can also drive an elevation in oxidative stress. Fibronectin (FN) is a major constituent of the extracellular matrix of PDA that can mediate integrin-induced reactive oxygen species (ROS) production by stromal cells. Thus oxidation therapy is an attractive strategy that takes advantage of the elevated level of oxidative stress typically present in solid tumors, especially PDA. We demonstrated previously that the matricellular protein Fibulin5 (Fbln5) reduces FN-mediated integrin-induced ROS production by competing with FN for binding to α5β1 integrin. Fbln5 binds to integrin via an RGD domain but does not activate integrin-induced ROS. Thus Fbln5 functions as a rheostat to dampen integrin-mediated ROS production. Mutation of the RGD domain in Fbln5 to RGE abolishes the binding of Fbln5 with the integrin. Here, we generated KIC (Fbln5+/+, LSL-KrasG12D, Ink4a/ArfLox/Lox, p48-Cre) and EE (Fbln5RGE/RGE) KIC mice to further explore the therapeutic potential of Fbln5 and underlying mechanisms. EEKIC mice survived longer and had smaller tumors than KIC littermates. Accordingly, ROS levels were higher in tumors from EEKIC mice. Importantly, survival and tumor burden could be returned to wild-type levels in EEKIC mice by systemic treatment with the antioxidant N-acetyl cysteine. ROS induction correlated with reduction in blood vessel density and endothelial cell proliferation in EEKIC tumors. Furthermore, gemcitabine was more effective in EEKIC mice. These data confirm the essential contribution of elevated ROS to disease burden in the EEKIC mice. To explore the oxidative pathways affected by the EE mutation in Fbln5, primary mouse embryonic fibroblasts (MEFs) and tumor-associated fibroblast (TAFs) were evaluated by qPCR gene array. The levels of multiple oxidative stress-induced genes were induced significantly in EE MEFs when plated on FN. These genes are being investigated for potential mechanistic contribution to the elevated ROS. To determine if Fbln5 is a target that can be exploited therapeutically, we developed a specific Fbln5-targeting peptoid (JM-454). Peptoids closely resemble peptides except that the side chains extend from the main chain nitrogen rather than the α-carbon. They are achiral, protease resistant, non-immunogenic and more cell/tissue permeable. JM-454 induces ROS both in MEFs plated on FN and orthotopic pancreatic tumors. The therapeutic efficacy of JM-454 will be examined later. Our studies show that a therapeutic antagonism of Fbln5 has the potential to be a first in class drug that harnesses the biology of ECM-integrin signaling to elevate oxidative stress in the tumor microenvironment. Citation Format: Miao Wang, Mary Topalovski, Jaya Matharage, Jason Toombs, Christopher Wright, Gomika Udugamasooriya, Rolf A. Brekken. Fibulin5 as a novel therapeutic target for pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4854. doi:10.1158/1538-7445.AM2014-4854

Split-and-pool synthesis and characterization of peptide tertiary amide library.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both α-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

1H NMR and theoretical studies on the conformational equilibrium of tryptophan methyl ester

Abstract Selected 3JHH coupling constants and theoretical calculations were used to explain the conformational equilibrium of L-tryptophan methyl ester (Trp-OMe) in several solvents. The obtained 3JHαHβ values did not exhibit any significant variability and thus indicate that there are no conformational population variations for the side chain of the Trp-O-Me depending on the solvent. Moreover, the potential energy surfaces obtained at the B3LYP/cc-pVDZ theoretical level produced eight energy minima that were analysed by QTAIM and NBO methods. It was possible to conclude that the Trp-OMe conformational preferences were due to hyperconjugative effects involving the nonbonding electron pairs of the main chain nitrogen atom and certain antibonding orbitals ( σ C 4 -C 13 ⁎ , σ C 1 -C 4 ⁎ and σ C 4 -H 12 ⁎ ) and also to the steric effects from the nonbonding electron pairs of oxygen atoms and the main and side chain of this system.

Structure Analysis and Comparative Characterization of the Cytochrome c′ and Flavocytochrome c from Thermophilic Purple Photosynthetic Bacterium Thermochromatium tepidum

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.

A possibility of a protein-bound water molecule as the ionizable group responsible for pKe at the alkaline side in human matrix metalloproteinase 7 activity

Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline pK(a) (pK(e1) and pK(e2)) values of about 4 and 10. The ionizable group for pK(e2) was assigned to Lys or Arg by thermodynamic analysis; however, no such residues are present in the active site. Hence, based on the crystal structure, we hypothesized that a water molecule bound to the main-chain nitrogen of Ala162 (W1) or the main-chain carbonyl oxygen of Pro217 (W2) is a candidate for the ionizable group for pK(e2) [Takeharu, H. et al. (2011) Biochim. Biophys. Acta 1814, 1940-1946]. In this study, we inspected this hypothesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), all 19 variants, in which one of all Lys and Arg residues was replaced by Ala, retained activity, indicating that neither Lys nor Arg is the ionizable group. pK(e2) values of A162S, A162V and A162G were 9.6 ± 0.1, 9.5 ± 0.1 and 10.4 ± 0.2, respectively, different from that of wild-type MMP-7 (WT) (9.9 ± 0.1) by 0.3-0.5 pH unit, and those of P217S, P217V and P217G were 10.1 ± 0.1, 9.8 ± 0.1 and 9.7 ± 0.1, respectively, different from that of WT by 0.1-0.2 pH unit. These results suggest a possibility of W1 or W2 as the ionizable group for pK(e2).

Structural insight into mechanism and diverse substrate selection strategy of L‐ribulokinase

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k(cat) values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptide chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified (447)GGLPQK(452) as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.

Abstract 2786: Identification and preclinical characterization of EMD 1204831 – A selective c-Met kinase inhibitor in clinical phase 1

Abstract The involvement of the mesenchymal endothelial transition factor (c-Met) in the primary event of oncogenic transformation and the secondary ability to mediate metastatic spread has been convincingly demonstrated in preclinical and early clinical settings. The clinical benefits of c-Met kinase inhibitors with various modes of actions and selectivity profiles are currently under investigation hoping that inhibitors of c-Met might emerge as valuable cancer therapeutics in the future. During an HTS run 3-(diethylamino)propyl N-[3-[[5-(3,4-dimethoxyphenyl)-2-oxo-6H-1,3,4-thiadiazin-3-yl]methyl]phenyl]carbamate was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): >100 µg/ml, IC50 (cMet enzyme): 30 nM, IC50 (cMet A549): 800 nM) providing a valid starting point. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting with the main chain nitrogen atom of Met1160 within the hinge region and with the main chain nitrogen of Asp1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1204831 was identified as development compound. EMD1204831 is currently being investigated in a phase 1 clinical trial. The pyridazinone EMD1204831 inhibits enzymatic and cellular c-Met kinase activity with IC50 values of 12 nM and 15 nM, respectively. EMD1204831 displayed an exquisite selectivity when tested in vitro against a panel of more than 400 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes. EMD 1204831 demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models, including U87-MG glioblastoma cells (autocrine HGF expression), TPR-Met-transformed mouse fibroblasts (oncogenic Met fusion protein) or Hs746T gastric cancer cells (c-Met gene amplification and HGF-independent activation). Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. PK/PD analysis revealed efficient, dose- and time-dependent inhibition of c-Met phosphorylation, reduction of IL-8 and cyclin D1 expression as well as an induction of the cell cycle inhibitor p27. The overall profile of EMD1204831 including first time structural disclosure, some structure activity relationships, in vitro potency, selectivity profile and in-vivo data will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2786. doi:10.1158/1538-7445.AM2011-2786

Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection

The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3β conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.

Structural and Mutational Analysis of Canonical Loop Residues In Protease Nexin 2 Involved In Factor XIa and Trypsin Inhibition.

AbstractAbstract 1147Factor XIa (FXIa) activates FIX and is regulated by platelet-secreted protease nexin 2 (PN2) that contains a Kunitz-type protease inhibitor (KPI) domain. Trypsin is regulated by basic pancreatic trypsin inhibitor (BPTI). The primary and tertiary structures of trypsin and the catalytic domain of FXIa are highly homologous, and KPI and BPTI are nearly identical structurally. We have previously identified two loop structures (loops 1 and 2) in the KPI domain of PN2 that interact with residues in the FXIa catalytic domain. Based on the structure of the FXIa/KPI complex crystal structure, residues within loops 1 and 2 were mutated for experiments examining the inhibition of FXIa and trypsin.Results show that the loop-1-region P1 site residue Arg15 of PN2KPI plays a major role in FXIa inhibition by protruding into the S1 specificity pocket of FXIa. Ala mutation at this site renders PN2KPI non-inhibitory for both FXIa and trypsin. BPTI has Lys15 at the P1 site. BPTI inhibits both FXIa and trypsin significantly less effectively than PN2KPI. PN2KPI-R15K lost FXIa inhibitory activity, whereas BPTI-K15R substantially gained affinity for FXIa. Like FXIa, trypsin preferred BPTI-K15R showing a significant enhancement in affinity. Thus, a major determinant of the inhibitory activity of PN2KPI and BPTI against FXIa and trypsin is the P1 residue, with Arg being preferred over Lys for both inhibitors and both proteases. In addition, loop 1 residues Pro13 and Arg20 make important contributions to both FXIa and trypsin inhibition as demonstrated by significantly elevated Ki values for Ala mutations (P13A, and R20A) at these sites. In contrast, Ser19 makes no significant contribution to inhibition of either FXIa or trypsin whereas Met17 makes a significant contribution to the inhibition of trypsin, but not FXIa. In loop 2, only Phe34 is identified as a residue making significant contributions to the inhibition of both FXIa and trypsin, since the PN2KPI-F34A mutant displayed reduced inhibitory activity for both FXIa (6-fold) and for trypsin (3-fold). To rationalize these findings, we examined the crystal structures of the FXIa(catalytic domain)/PN2KPI complex, and the trypsin/BPTI complex. Structurally, the PN2KPI loop-1, P1-site residue Arg15 makes a complex primary interaction with Asp189 of both FXIa and trypsin. Disruption of this site by R15A mutation renders PN2KPI non-inhibitory because it preempts salt bridge interactions from two nitrogen atoms of the guanidinium group of Arg15 with Asp189 and Gly218 in FXIa. In addition, the Arg15 carbonyl oxygen forms hydrogen bonds with main-chain nitrogen atoms of one of the catalytic triad residues, Ser195, and with Gly193. The other important interaction in FXIa/PN2KPI or trypsin/BPTI is hydrophobic, between PN2KPI-Phe34 and FXIa-Tyr143 and between BPTI-Val34 and trypsin-Tyr151. This intermolecular interaction is further strengthened by an intramolecular interaction in which the side chain of Phe34 packs closely with the side chain of Met17 within PN2KPI, altogether forming a strong hydrophobic patch in FXIa-PN2KPI and trypsin-PN2KPI. PN2KPI-F34A disrupts both inter- and intramolecular hydrophobic interactions, leading to discernable reductions in affinity for both FXIa and trypsin. Despite occupying extreme positions in the autolysis loops, (143YRKLRDKI151 in FXIa and 143NTKSSGTSY151 in trypsin), Tyr143/151 residues still orient themselves in close proximity to Phe34. Thus, loop-1 residues of PN2KPI establish complex ionic interactions that play a major role, which is supplemented by the loop-2 residue, Phe34 (in PN2KPI) or Val34 (in BPTI), which establish hydrophobic interactions with residues in FXIa and trypsin leading to very high-affinity enzyme-inhibitor complexes. Disclosures:No relevant conflicts of interest to declare.

ChemInform Abstract: Conformational Regulation of Amyloid β‐Peptide by Lipid Membranes and Metal Ions

AbstractReview: 28 refs.

Structure of Cinaciguat (BAY 58–2667) Bound to Nostoc H-NOX Domain Reveals Insights into Heme-mimetic Activation of the Soluble Guanylyl Cyclase

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

Abstract 5777: EMD 1214063, an exquisitely selective c-Met kinase inhibitor in clinical phase 1

Abstract The role of the receptor tyrosin kinase c-Met in tumor progression, metastasis and aggressiveness has been convincingly demonstrated in preclinical and early clinical settings. Several compounds with different selectivity profiles inhibiting c-Met are currently under preclinical/clinical investigation and might emerge as valuable cancer therapeutics in the future. During an HTS run N-(3-(3,6-Dihydro-5-(3,4-dimethoxyphenyl)-2-oxo-2H-1,3,4-thiadiazin-3-ylmethyl)-phenyl)-carbaminic acid-(3-(N,N-diethylamino)-propylester) (1) was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): >100 µg/ml, IC50 (c-Met in vitro): 30 nM, IC50 (c-Met cellular): 800 nM) providing a valid starting point for lead optimization. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting namely with the main chain nitrogen atom of methionine 1160 within the hinge region and with the main chain nitrogen of aspartic acid 1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1214063 was identified as candidate for further development and is currently investigated in a phase 1 clinical trial. The pyridazinone EMD1214063 inhibits enzymatic and cellular c-Met kinase activity with IC50 values in the low nanomolar range. This compound displayed an exquisite selectivity when tested in vitro against a panel of more than 250 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes and demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models. Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. The overall profile of EMD1214063 including first time disclosure of the precise structure, synthesis, structure activity relationships, in vitro potency, selectivity profile, pharmacokinetic and in vivo data will be discussed. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5777.

脂質膜と金属イオンによるアミロイドβペプチドのコンホメーション制御

Conformational transition of monomeric amyloid beta-peptide (Abeta) to a self-associated beta-sheet structure is considered to be an initial step in the development of Alzheimer's disease. Several lines of evidence suggest that physiologically abundant lipid membranes and metal ions are involved in this step. We have demonstrated that Abeta binds to the phosphatidylcholine membrane in the lamellar gel phase but not in the liquid crystalline phase by using fluorescence and circular dichroism spectroscopy. The membrane-bound Abeta molecule takes alpha-helical or beta-sheet structure depending on the temperature. Tightly packed phosphatidylcholine membranes appear to serve as a platform for non-electrostatic binding and self-association of Abeta. We have also examined Zn(II) and Cu(II) binding modes of Abeta by Raman spectroscopy. The Raman spectra demonstrate that three histidine residues in the N-terminal region of Abeta provide primary metal binding sites. Zn(II) binds to the N(tau) atom of histidine and the peptide aggregates through intermolecular His-Zn-His bridges. In contrast, Cu(II) ion is chelated by the N(pi) atom of histidine and deprotonated main-chain amide nitrogens to form a soluble complex. Our findings on the conformational regulation of Abeta may help in better understanding the molecular basis for the development of Alzheimer's disease.