Redesign of protein domains using one-bead-one-compound combinatorial chemistry

A novel combinatorial strategy for the redesign of proteins based on the strength and specificity of intra- and interprotein interactions is described. The strategy has been used to redesign the hydrophobic core of the B domain of protein A. Using one-bead-one-compound combinatorial chemistry, 300 analogues of the C-terminal alpha-helix of the B domain, H3x, have been synthesized using a biocompatible resin and the HMFS linker, allowing the screening to occur while the peptides were bound to the resin. The screening was based on the ability of the H3x analogues to interact with the N-terminal helices of the B domain, H1-H2, and retain the native B domain activity, that is binding to IgG. Eight different analogues containing some nonconservative mutations were obtained from the library, the two most frequent of which, H3P1 and H3P2, were studied in detail. CD analysis revealed that the active analogues interact with H1-H2. To validate the redesign strategy the covalent modified domains H1-H2-H3P1 and H1-H2-H3P2 were synthesized and characterized. CD and NMR analysis revealed that they had a unique, stable, and well-defined three-dimensional structure similar to that for the wild-type B domain. This combinatorial strategy allows us to select for redesigned proteins with the desired activity or the desired physicochemical properties provided the right screening test is used. Furthermore, it is rich in potential for the chemical modification of proteins overcoming the drawbacks associated with the total synthesis of large protein domains.     

Dinuclear zinc(II) complexes of symmetric Schiff-base ligands with extended quinoline sidearms

The synthesis of two 2-formylquinolines is reported via the Skraup method followed by SeO(2) oxidation. Each aldehyde is condensed with (1R,2R)-diaminocyclohexane and (R)-BINAM, yielding four enantiomerically-pure bis(imine-quinoline) ligands. The neutral ligands are reacted with ZnCl(2) to give complexes with bis(bidentate) coordination of ZnCl(2) units. X-Ray structural characterization of three complexes shows them to have a single-stranded helical motif, with M helicity, except in one case where a 1:1 mixture of M and P helices is seen. The ligands and complexes are further characterized spectroscopically by solution (1)H and (13)C NMR, UV-vis and ECD.     

Structure-activity relationships of GHRP-6 azapeptide ligands of the CD36 scavenger receptor by solid-phase submonomer azapeptide synthesis

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W(2)]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F(4)]GHRP-6, 2, exhibited a characteristic β-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 μM) affinity for the CD36 scavenger receptor.     

Structure-based screening as applied to human FABP4: a highly efficient alternative to HTS for hit generation

The time-limiting step in HTS often is the development of an appropriate assay. In addition, hits from HTS fairly often turn out to be false positives and generally display unfavorable properties for further development. Here we describe an alternative process for hit generation, applied to the human adipocyte fatty acid binding protein FABP4. A small molecular ligand for FABP4 that blocks the binding of endogenous ligands may be developed into a drug for the treatment of type-2 diabetes. Using NMR spectroscopy, we screened FABP4 for low-affinity binders in a diversity library consisting of small soluble scaffolds, which yielded 52 initial hits in total. The potencies of these hits were ranked, and crystal structures of FABP4 complexes for two of the hits were obtained. The structural data were subsequently used to direct similarity searches for available analogues, as well as chemical synthesis of 12 novel analogues. In this way, a series of three selective FABP4 ligands with attractive pharmacochemical profiles and potencies of 10 microM or better was obtained.     

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.     

Sequence-specific recognition and cooperative dimerization of N-terminal aromatic peptides in aqueous solution by a synthetic host

This article describes the selective recognition and noncovalent dimerization of N-terminal aromatic peptides in aqueous solution by the synthetic host compound, cucurbit[8]uril (Q8). Q8 is known to bind two aromatic guests simultaneously and, in the presence of methyl viologen, to recognize N-terminal tryptophan over internal and C-terminal sequence isomers. Here, the binding of Q8 to aromatic peptides in the absence of methyl viologen was studied by isothermal titration calorimetry (ITC), (1)H NMR spectroscopy, and X-ray crystallography. The peptides studied were of sequence X-Gly-Gly, Gly-X-Gly, and Gly-Gly-X (X = Trp, Phe, Tyr, and His). Q8 selectively binds and dimerizes Trp-Gly-Gly (1) and Phe-Gly-Gly (4) with high affinity (ternary K = 10(9)-10(11) M(-)(2)); binding constants for the other 10 peptides were too small to be measured by ITC. Both peptides bound in a stepwise manner, and peptide 4 bound with positive cooperativity. Crystal structures of Q8.1 and Q8.4(2) reveal the basis for selective recognition as simultaneous inclusion of the hydrophobic aromatic side chain into the cavity of Q8 and chelation of the proximal N-terminal ammonium group by carbonyl groups of Q8. The peptide sequence selectivity and positively cooperative dimerization reported here are, to the best of our knowledge, unprecedented for synthetic hosts in aqueous solution. Specific peptide recognition and dimerization by synthetic hosts such as Q8 should be important in the study of dimer-mediated biochemical processes and for the separation of peptides and proteins.     

Investigating the hydrogen-bond acceptor site of the nicotinic pharmacophore model: a computational and experimental study using epibatidine-related molecular probes

The binding mode of nicotinic agonists has been thoroughly investigated in the last decades. It is now accepted that the charged amino group is bound by a cation-π interaction to a conserved tryptophan residue, and that the aromatic moiety is projected into a hydrophobic pocket deeply located inside the binding cleft. A hydrogen bond donor/acceptor, maybe a water molecule solvating this receptor subsite, contributes to further stabilize the nicotinic ligands. The position of this water molecule has been established by several X-ray structures of the acetylcholine-binding protein. In this study, we computationally analyzed the role of this water molecule as a putative hydrogen bond donor/acceptor moiety in the agonist binding site of the three most relevant heteromeric (α4β2, α3β4) and homomeric (α7) neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Our theoretical investigation made use of epibatidine 1 and deschloroepibatidine 2 as molecular probes, and was then extended to their analogues 3 and 4, which were subsequently synthesized and tested at the three target receptor subtypes. Although the pharmacological data for the new ligands 3 and 4 indicated a reduction of the affinity at the studied nAChRs with respect to reference agonists, a variation of the selectivity profile was clearly evidenced.     

Coordination driven self-assembly of four new molecular boats using a flexible imidazole-containing donor linker

Using a metal-ligand coordination bonding approach, the self-assembly of four new metallamacrocycles from Pd(ii)-based 90 degrees acceptors and a diimidazole donor ligand 1,3-bis(imidazole-1-ylmethyl)-2,4,6-trimethylbenzene (L) has been achieved. The assemblies are characterized fully by NMR and electrospray ionization-mass spectroscopic (ESI-MS) analysis and in two cases the X-ray single-crystal structure analysis established the gross structures. The selective formation of a diimidazole-based linker (L) containing macrocycle [(en)Pd(micro-L)2Pd(en)]4+ from a 1 : 1 : 1 mixture of cis-Pd(en)(NO3)2, and 1,2-bis(4-pyridyl)ethane is also established. Measuring the binding constants established the stronger Pd- binding force compared to traditional Pd-N(pyridyl linker) interaction, which reveals the possibility of using imidazole donor ligands as potential linkers or even better ligands compared to the widely used pyridyl donor ligands in the construction of metal-based large supramolecular assemblies.     

含(1-芳乙酰胺基-2-叔胺基)乙烷结构的六氢-1H-1,4-二氮(艹卓)类化合物的合成与镇痛活性

2-或5-取代的六氢-1H-1,4-二氮(艹卓)类化合物(1～3)经单酰化及酰化反应后,合成了16个带有(1-芳乙酰胺基-2-叔氨基)乙烷结构的六氢-1H-1,4-二氮(艹卓)类目标化合物(5～9,11～13,15～17,19～23),经元素分析、IR、MS和¹H NMR确证了其组成和结构。对所有目标化合物都进行了豚鼠回肠试验,初步药理试验表明,16个化合物对受试标本显示不同程度的抑制作用,对抑制率较高的两个化合物5和7测试了IC₅₀值。对在豚鼠回肠试验中显示较强激动作用的4个化合物进行了小鼠扭体法镇痛活性试验,测得了其ED₅₀值。     

Binding of Ni2+ and Cu2+ ions to peptides with a Cys-His motif

Waglerin I is a 22 amino acid snake venom toxin. Its three fragments (GGKPDLRPCHP-NH2, PCHYIPRPKPR-NH2, PCHPPCHYIPR-NH2), due to the presence of two Cys and His residues, are potentially very attractive ligands for transition metal ions. The main aim of this work was to establish the impact of these two adjacent residues on Ni2+ ion binding, especially because this kind of motif is very common in nature, and the study of low molecular weight models could be helpful in understanding larger systems. In this work waglerin fragments and their N-protected analogues were studied with Ni2+ (and Cu2+ for peptides with disulfide bridges) ions using combined potentiometric and spectroscopic measurements (UV-Vis, CD, EPR and NMR). In all peptides, except PCHPPCHYIPR-NH2 with a disulfide bridge, the Cys-His motif was found to be crucial for the coordination of Ni2+ ions. In the case of the N-unprotected analogues, the N-terminal amino group participates in the coordination as well.     

Stachyodin A,a pterocarpan derivative with unusual spirotetrahydrofuran ring from the roots of Indigofera stachyodes

Stachyodin A (1), a rearrangement product of pterocarpan featuring a 6/5/5/6 tetracyclic ring system with an unusual spirotetrahydrofuran ring, and stachyodin B (2), a new dihydrochalcone, along with five known analogues (3–7) were isolated from the roots of Indigofera stachyodes. The structures of 1 and 2 were elucidated on the basis of their HRESIMS and NMR spectroscopic data, and their absolute configurations were determined by X-ray crystallographic analysis and electronic circular dichroism (ECD) data, respectively. Compounds 2–4 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells.     

Distributed computing and NMR constraint-based high-resolution structure determination: applied for bioactive Peptide endothelin-1 to determine C-terminal folding

Distributed computing has been implemented to the solution structure determination of endothelin-1 to evaluate efficiency of the method for NMR constraint-based structure calculations. A key target of the investigation was determination of the C-terminal folding of the peptide, which had been dispersed in previous studies of NMR, despite its pharmacological significances. With use of tens of thousands of random initial structures to explore the conformational space comprehensively, we determined high-resolution structures with good convergences of C-terminal as well as previously defined N-terminal structures. The previous studies had missed the C-terminal convergence because of initial structure dependencies trapped in localized folding of the N-terminal region, which are strongly constricted by two disulfide bonds.     

Exploring the Subtle Effect of Aliphatic Ring Size on Minor Actinide-Extraction Properties and Metal Ion Speciation in Bis-1,2,4-Triazine Ligands

The synthesis and evaluation of three novel bis-1,2,4-triazine ligands containing five-membered aliphatic rings are reported. Compared to the more hydrophobic ligands 1 – 3 containing six-membered aliphatic rings, the distribution ratios for relevant f-block metal ions were approximately one order of magnitude lower in each case. Ligand 10 showed an efficient, selective and rapid separation of Am^III and Cm^III from nitric acid. The speciation of the ligands with trivalent f-block metal ions was probed using NMR titrations and competition experiments, time-resolved laser fluorescence spectroscopy and X-ray crystallography. While the tetradentate ligands 8 and 10 formed Ln^III complexes of the same stoichiometry as their more hydrophobic analogues 2 and 3 , significant differences in speciation were observed between the two classes of ligand, with a lower percentage of the extracted 1:2 complexes being formed for ligands 8 and 10 . The structures of the solid state 1:1 and 1:2 complexes formed by 8 and 10 with Y^III, Lu^III and Pr^III are very similar to those formed by 2 and 3 with Ln^III. Ligand 10 forms Cm^III and Eu^III 1:2 complexes that are thermodynamically less stable than those formed by ligand 3 , suggesting that less hydrophobic ligands form less stable An^III complexes. Thus, it has been shown for the first time how tuning the cyclic aliphatic part of these ligands leads to subtle changes in their metal ion speciation, complex stability and metal extraction affinity.     

Abiotic metallofoldamers as electrochemically responsive molecules

Described are the design, synthesis, and study of nonbiological molecules based on salophen and salen ligands that fold into single-stranded helices in the presence of either Ni(II) or Cu(II). X-ray diffraction studies show that the materials fold into helical structures in the solid state, and a series of NMR studies provide strong evidence that the folded structures are conserved in solution. Metal coordination is required for folding, as NMR and X-ray show that the free ligands do not adopt helical structures. Two of the racemic metallofoldamers spontaneously resolve during crystallization from CHCl3/acetonitrile, and CD spectroscopy and optical rotation show that the resolved, crystalline materials racemize quickly when dissolved at 5 degrees C. This shows that the secondary structures can reorganize easily and can, therefore, provide the basis for responsive materials. By comparison, an analogue from enantiomerically pure (R,R)-(-)-trans-cyclohexanediamine showed a strong CD signal and a large specific rotation. Electrochemical experiments show that a structural reorganization occurs upon metal-centered reduction of a Cu(II)-containing foldamer. When the reduction is carried out in the presence of coordinating ligands, it is proposed that apical binding of those ligands gives square pyramidal complexes. Semiempirical (AM1) calculations support that the helical structure would be disrupted by the reduction to Cu(I) with concomitant reorganization to a square pyramidal complex.     

Chiroptical Detection of Nonchromophoric,Achiral Guests by Enantiopure Alleno‐Acetylenic Helicages

Enantiopure alleno‐acetylenic ligands assemble diastereoselectively upon the addition of a zinc(II) salt to form triple‐stranded helicates, which provide a sufficiently large helical cage (“helicage”) for the encapsulation of guests. The inclusion complexation of heteroalicycles is confirmed by ROESY and DOSY NMR spectroscopy and quantified in ¹H NMR binding titrations. The ECD spectra of the helicates, which showed strong Cotton effects and exciton coupling, were found to be extremely sensitive to the nature of the guest molecules. Consequently, a series of nonchromophoric, achiral guests of different sizes as well as regioisomers (1,3‐ and 1,4‐dioxane) became distinguishable on the basis of their induced CD (ICD) spectra. Molecular dynamics (MD) simulations show the adaptability of the cavity size to individual guest molecules and support the selective ICD output. Particularly high affinity towards 1,4‐dioxane allowed its selective detection at parts‐per‐million (ppm) levels in aqueous solutions.     

Structural Heterogeneity of Doubly-Charged Peptide b-Ions

Performing collisionally activated dissociation (CAD) and electron capture dissociation (ECD) in tandem has shown great promise in providing comprehensive sequence information that was otherwise unobtainable by using either fragmentation method alone or in duet. However, the general applicability of this MS³ approach in peptide sequencing may be undermined by the formation of non-direct sequence ions, as sometimes observed under CAD, particularly when multiple stages of CAD are involved. In this study, varied-sized doubly-charged b-ions from three tachykinin peptides were investigated by ECD. Sequence scrambling was observed in ECD of all b-ions from neurokinin A (HKTDSFVGLM-NH₂), suggesting the presence of N- and C-termini linked macro-cyclic conformers. On the contrary, none of the b-ions from eledoisin (pEPSKDAFIGLM-NH₂) produced non-direct sequence ions under ECD, as it does not contain a free N-terminal amino group. ECD of several b-ions from Substance P (RPKPQQFFGLM-NH₂) showed series of c_m-Lys fragment ions which suggested that the macro-cyclic structure may also be formed by connecting the C-terminal carbonyl group and the ε-amino group of the lysine side chain. Theoretical investigation of selected Substance P b-ions revealed several low energy conformers, including both linear oxazolones and macro-ring structures, in corroboration with the experimental observation. This study showed that a b-ion may exist as a mixture of several forms, with their propensities influenced by its N-terminus, length, and certain side-chain groups. Further, the presence of several macro-cyclic structures may result in erroneous sequence assignment when the combined CAD and ECD methods are used in peptide sequencing.     

Chiral spiro Cu(I) complexes. Supramolecular stereocontrol and isomerisation dynamics by the use of TRISPHAT anions

Association of enantiopure TRISPHAT anion (1) with chiral spiro [Cu(LL')2] complexes (LL' = 2-R-phen, 2, 6-R-bpy, 3, and 2-iminopyridine, 4) leads to an efficient NMR enantiodifferentiation. Variable temperature 1H NMR spectroscopy has been used to determine the isomerisation kinetics of these pseudo-tetrahedral complexes and to evaluate their configurational stability; the latter depending on the structure of the diimine ligands. In the case of the 2-anthracenyl-phen derivative, a decent level of supramolecular stereocontrol was noted (d.e. up to 45%); the configuration of the complex being determined by electronic circular dichroism (ECD).     

Concise Synthesis and Cellular Evaluation of 3′-Formyl-4′,6′-dihydroxy-2′-methoxy-5′-methylchalcone (FMC) and Its Analogues

3′-Formyl-4′,6′-dihydroxy-2′-methoxy-5′-methylchalcone (FMC) was a natural product isolated from Cleistocalyx operculatus. A four-step synthetic strategy toward FMC and its four analogues (1b–1e) was first developed. All compounds were synthesized from commercially available 2,4,6-trihydroxyacetophenone; formylation at 3′ position under Vilsmeier–Haack conditions was followed by the introduction of a methyl group at 5′ position. The key step of selective methylation at 2′ position was achieved by trimethylsilyldiazomethane (TMSCHN₂). Then substituted aromatic aldehydes were condensed through Claisen–Schmidt reaction in the presence of potassium hydroxide. All structures were confirmed by ¹H NMR, ¹³C NMR, and high-resolution mass spectra. FMC and analogues were screened for their antiproliferative activity.     

A reactivity pattern for discrimination of ER agonism and antagonism based on 3-D molecular attributes

Various models have been developed to predict the relative binding affinity (RBA) of chemicals to estrogen receptors (ER). These models can be used to prioritize chemicals for further tiered biological testing to assess the potential for endocrine disruption. One shortcoming of models predicting RBA has been the inability to distinguish potential receptor antagonism from agonism, and hence in vivo response. It has been suggested that steroid receptor antagonists are less compact than agonists; thus, ER binding of antagonists may prohibit proper alignment of receptor protein helices preventing subsequent transactivation. The current study tests the theory of chemical bulk as a defining parameter of antagonism by employing a 3-D structural approach for development of reactivity patterns for ER antagonists and agonists. Using a dataset of 23 potent ER ligands (16 agonists, 7 antagonists), molecular parameters previously found to be associated with ER binding affinity, namely global ( E HOMO ) and local (donor delocalizabilities and charges) electron donating ability of electronegative sites and steric distances between those sites, were found insufficient to discriminate ER antagonists from agonists. However, parameters related to molecular bulk, including solvent accessible surface and negatively charged Van der Waal's surface, provided reactivity patterns that were 100% successful in discriminating antagonists from agonists in the limited data set tested. The model also shows potential to discriminate pure antagonists from partial agonist/antagonist structures. Using this exploratory model it is possible to predict additional chemicals for their ability to bind but inactivate the ER, providing a further tool for hypothesis testing to elucidate chemical structural characteristics associated with estrogenicity and anti-estrogenicity.     

Structural basis for the activation of platelet integrin αIIbβ3 by calcium- and integrin-binding protein 1

Calcium and integrin binding protein 1 (CIB1) is a specific binding partner for the cytoplasmic domain of the αIIb subunit of the highly abundant platelet integrin αIIbβ3. This protein has been suggested to be involved in the regulation of the activation of αIIbβ3, a process leading to platelet aggregation and blood coagulation. In this work, the solution structure of the deuterated Ca(2+)-CIB1 protein complexed with an αIIb peptide was first determined through modern RDC-based NMR methods. Next, we generated a complex structure for CIB1 and the αIIb domain (Ca(2+)-CIB1/αIIb) using the program Haddock, which is based on experimental restraints obtained for the protein-peptide interface from cross-saturation NMR experiments. In this data-driven complex structure, the N-terminal α-helix of the cytoplasmic domain of αIIb is buried in the hydrophobic pocket of the C-lobe of Ca(2+)-CIB1. The C-terminal acidic tail of αIIb remains unstructured and likely interacts with several positively charged residues in the N-lobe of Ca(2+)-CIB1. A potential molecular mechanism for the CIB1-mediated activation of the platelet integrin could be proposed on the basis of the model structure of this protein complex. Another feature of this work is that, in the NMR cross-saturation experiments, we applied the selective radio frequency irradiation to the smaller binding partner (the αIIb peptide), and successfully detected the binding interface on the larger binding partner Ca(2+)-CIB1 through its selectively protonated methyl groups. This 'reverse' methodology has a broad potential to be employed to many other complexes where synthetic peptides and a suitably isotope-labeled medium- to large-sized protein are used to study protein-protein interactions.