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The structure-based design of small-molecule inhibitors targeting protein–protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.

RAS proteins are central in the proliferation of many types of cancer, but a general approach toward the identification of pan-mutant RAS inhibitors has remained unresolved. In this work, we describe the application of a binding pharmacophore identified from analysis of known RAS binding peptides to the design of novel peptides. Using a chemically divergent approach, we generated a library of small stapled peptides from which we identified compounds with weak binding activity. Exploration of structure-activity relationships (SARs) and optimization of these early compounds led to low-micromolar binders of KRAS that block nucleotide exchange.

An efficient macrocyclisation approach based on the double aromatic nucleophilic substitution (SNACK) was developed. This methodology allows a facile incorporation of heterocyclic motifs into macrocyclic rings and rapid synthesis of a significant number of structurally diverse macrocycles. SNACK macrocyclisation enables preparation of stable diastereoisomers of conformationally restricted macrocycles (atropisomers). Practical application of SNACK macrocyclisation in a drug discovery project was exemplified by the identification of high affinity macrocyclic binders of B-cell lymphoma 6 (BCL6).

"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.

The conformational behavior of a small molecule free in solution is important to understand the free energy of binding to its target. This could be of special interest for proteolysis-targeting chimeras (PROTACs) due to their often flexible and lengthy linkers and the need to induce a ternary complex. Here, we report on the molecular dynamics (MD) simulations of two PROTACs, MZ1 and dBET6, revealing different linker conformational behaviors. The simulation of MZ1 in dimethyl sulfoxide (DMSO) agrees well with the nuclear magnetic resonance study, providing strong support for the relevance of our simulations. To further understand the role of linker plasticity in the formation of a ternary complex, the dissociation of the complex von Hippel-Lindau-MZ1-BRD4 is investigated in detail by steered simulations and is shown to follow a two-step pathway. Interestingly, both MZ1 and dBET6 display in water, a tendency toward an intramolecular lipophilic interaction between the two warheads. The hydrophobic contact of the two warheads would prevent them from binding to their respective proteins and might have an effect on the efficacy of induced cellular protein degradation. However, conformations featuring this hydrophobic contact of the two warheads are calculated to be marginally more favorable.

International audience; Efficient drug discovery is based on a concerted effort in optimizing bioactivity and compound properties such as lipophilicity, and is guided by efficiency metrics that reflect both aspects. While conformation–activity relationships and ligand conformational control are known strategies to improve bioactivity, the use of conformer-specific lipophilicities (logp) is much less explored. Here we show how conformer-specific logp values can be obtained from knowledge of the macroscopic logP value, and of the equilibrium constants between the individual species in water and in octanol. This is illustrated with fluorinated amide rotamers, with integration of rotamer 19F NMR signals as a facile, direct method to obtain logp values. The difference between logp and logP optimization is highlighted, giving rise to a novel avenue for lipophilicity control in drug discovery.

An efficient macrocyclisation approach based on the double aromatic nucleophilic substitution (SNACK) was developed. This methodology allows a facile incorporation of heterocyclic motifs into macrocyclic rings and rapid synthesis of a significant number of structurally diverse macrocycles. SNACK macrocyclisation enables preparation of stable diastereoisomers of conformationally restricted macrocycles (atropisomers). Practical application of SNACK macrocyclisation in a drug discovery project was exemplified by the identification of high affinity macrocyclic binders of B-cell lymphoma 6 (BCL6).

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.

[Image: see text] Herein we report the use of metathesis to construct a novel tetracyclic core in a series of estrogen receptor degraders. This improved the chemical stability, as assessed using an NMR-MS based assay, and gave a molecule with excellent physicochemical properties and pharmacokinetics in rat. X-ray crystallography established minimal perturbation of the bridged compounds relative to the unbridged analogues in the receptor binding pocket. Unfortunately, despite retaining excellent binding to ERα, this adversely affected the ability of the compounds to degrade the receptor.

The three-dimensional conformations adopted by a free ligand in solution impact bioactivity and physicochemical properties. Solution 1D NMR spectra inherently contain information on ligand conformational flexibility and three-dimensional shape, as well as the propensity of the free ligand to fully preorganize into the bioactive conformation. Herein we discuss some key learnings, distilled from our experience developing potent and selective synthetic macrocyclic inhibitors, including Mcl-1 clinical candidate AZD5991. Case studies have been selected from recent oncology research projects, demonstrating how 1D NMR conformational signatures can complement X-ray protein-ligand structural information to guide medicinal chemistry optimization. Learning to extract free ligand conformational information from routinely available 1D NMR signatures has proven to be fast enough to guide medicinal chemistry decisions within design cycles for compound optimization.

Herein, we report the identification and synthesis of a series of tricyclic indazoles as a novel class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to the removal of a reactive metabolite signal in an in vitro glutathione trapping assay. Further optimization, guided by X-ray crystal structures and NMR conformational work, varied the alkyl side chain and pendant aryl group and resulted in compounds with low turnover in human hepatocytes and enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms of pharmacology and demonstrated the desired estrogen receptor α degrader-antagonist profile and demonstrated activity in a xenograft model of breast cancer.

Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRAS

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline

Gaining insight into the uptake of drugs into cells, trafficking and their target engagement enhances understanding of the drug’s function and efficiency. Here we study an antisense oligonucleotide drug (ASO) delivered into HEK293T and HeLa cells, by Nuclear Magnetic Resonance (NMR). Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by qRT-PCR, measuring downregulation of its target mSTAT3. Applying DNP NMR to frozen cells, we overcome limitations of traditional solution-state in-cell NMR (e.g. size, stability and sensitivity) as well as of visualization techniques, where (e.g. fluorescent) tagging of the ASO decreases its activity. The possibility to study an untagged, active drug, interacting in its natural environment, will increase insights into molecular mechanisms of delivery, intracellular trafficking and target engagement in intact cells.

B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.

The estrogen receptor alpha (ERα) is expressed in >70% of breast cancers and is a clinically validated target in oncology.1 Anti-hormonal therapies that directly block ER function (e.g., tamoxifen) or therapies that block the production of estrogen itself (e.g., aromatase inhibitors) have proven to be effective treatments of the disease. Further advances have been made with the development of SERDs (Selective Estrogen Receptor Degraders) such as fulvestrant which both antagonise ERα-driven tumor cell growth and cause degradation of the ERα receptor.2 We previously disclosed the identification of a tricyclic indole scaffold that led to the orally active clinical candidate AZD9496.3 Additional work to identify novel chemotypes including phenol 14 and indazole 25 was also disclosed together. The work previously disclosed relied on the presence of the acrylic acid for efficient degradation of the estrogen receptor. In this poster we will discuss the replacement of the acrylic acid on the tricyclic indole scaffold with amines (e.g. compound 3). Compound 3 is a degrader of the estrogen receptor in the MCF-7 cell line (pIC50 8.4). Further optimisation of lead structure 3 led to more potent selective degraders of the estrogen receptor in multiple cell lines (e.g. pIC50 >10 in MCF-7) with suitable properties for oral absorption (e.g. oral AUC 44 μM.h in mice at 20 mg/kg). We will discuss some of the medicinal chemistry challenges that were faced along the way and the optimisation strategy (use of NMR derived solution phase conformations, understanding of the binding mode in the estrogen receptor by X-ray crystallography). ol> Citation Format: Bernard Barlaam, Jason Breed, Rodrigo J Carbajo, Eric Gangl, Samantha Hughes, Christopher J Morrow, Thomas A Moss, Radoslaw Polanski, Willem M Nissink, Daniel O'Donovan, Jeffrey Varnes, Bin Yang, James S Scott. Small Molecule Degraders of the Estrogen Receptor (SERDs): Optimization of the tricyclic indole scaffold beyond AZD9496 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A107. doi:10.1158/1535-7163.TARG-19-A107

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

Heparanase is an endoglycosidase that specifically degrades heparan sulfate, one of the main components of the extracellular matrix. Heparanase is implicated in cancer processes such as tumour formation, angiogenesis and metastasis, making it a very attractive target in drug discovery. Its active form is a heterodimer constituted by a 45 kDa glycosylated subunit (Lys158-Ile543) non-covalently bound to a smaller 8 kDa polypeptide (Gln36-Glu109). Residues Glu225 and Glu343 are critical in its catalytic mechanism and two heparan sulfate binding sites (Lys158-Asp171 and Gln270-Lys280) have been identified in the enzyme. Here we report the (1)H, (13)C and (15)N chemical shift assignments, secondary structure and chemical shift deviations from random coil of the domain of human heparanase comprising residues Lys158-Lys417, a construct that has been validated as surrogate of the full length protein in the search of novel inhibitors for this enzyme.

AlfB and AlfC α-l-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3- N -acetylglucosamine and fucosyl-α-1,6- N -acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p -nitrophenyl-α- l -fucopyranoside into fucosyl-α-1,3- N -acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6- N -acetylglucosamine.

NMR analysis of four recombinant jerdostatin molecules was assessed to define the structural basis of two naturally occurring gain-of-function events: C-terminal dipeptide processing and mutation of the active residue K21 to arginine. Removal of the highly mobile and a bulky C-terminal dipeptide produced pronounced chemical shift changes in the sequentially unconnected but spatially nearby α1β1 inhibitory loop. Analysis of chemical shift divergence and 15N backbone relaxation dynamics indicated differences in motions in the picosecond to nanosecond time scale, and the higher T2 rate of S25, S26, and H27 of rJerK21 point to a slowdown in the microsecond to millisecond motions of these residues when compared with rJerR21. The evidence presented in this article converges on the hypothesis that dynamic differences between the α1β1 recognition loops of rJerR21 and rJerK21 may influence the thermodynamics of their receptor recognition and binding. A decrease in the μs-ms time scale may impair the binding affinity by reducing the rate of possible conformations that the rJerK21 can adopt in this time scale. Proteins 2011; © 2011 Wiley-Liss, Inc.

Sepsis is a leading cause of mortality that is most often provoked by endotoxins (i.e. lipopolysaccharides; LPS) released by Gram-negative bacteria into the patient's bloodstream during infection. The therapeutic armory currently available for sepsis treatment is poor. We previously identified an LPS-neutralizing small molecule, PTD7. Here we tested the efficacy of novel PTD7-nanoconjugates in a murine model of sepsis. We found that PTD7-based nanoconjugates treated mice had improved survival that it was correlated with a marked decrease in proinflammatory cytokines in the blood. This proves that nanoconjugate-based endotoxin neutralizers can function as intracorporeal neutralizers of bacterial endotoxins. (C) 2009 Elsevier B.V. All rights reserved.

Clinical proof of concept for polymer conjugates has already been achieved over the last 30 years, with a family of polymer-protein conjugates reaching the market and an exponentially growing list of polymer-drug conjugates currently in clinical trials. However, many challenges and opportunities still lie ahead, providing scope to develop this platform technology further.The delivery of new anticancer agents aimed at novel molecular targets and their combination, the development of both new polymeric materials with defined architectures and the treatment of diseases other than cancer are the most exciting and promising areas. The latest advances and future trends in the polymer conjugate field will be presented in this article, providing an insight into their potential in the clinics and offering a wide range of research approaches within the scientific community.Polymer therapeutics is a rapidly emerging field with exponentially growing opportunities to achieve medical treatments with highly enhanced therapeutic value.

Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (alpha-1,4 polyglucose, a natural polymer degraded by alpha-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9-32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34-69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with alpha-amylase led to reinstatement of activity to a maximum of 92-115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate (dextrin Mw = 47200 g/mol; MSH content 37 wt %), and its biological activity (+/-alpha-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of alpha-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The choice of masking polymer, activation mechanism, and the rate of unmasking can be tailored to therapeutic application.

The role of hydrophobic amino acids in the formation of hydrophobic cores as one of the major driving forces in protein folding has been extensively studied. However, the implication of neutral solvent-exposed amino acids is less clear and available information is scarce. We have used a combinatorial approach to study the structural relevance of three solvent-exposed residues (Tyr(327), Thr(329), and Gln(331)) located in thebeta-sheet of the tetramerization domain of the tumor suppressor p53 (p53TD). A conformationally defined peptide library was designed where these three positions were randomized. The library was screened for tetramer stability. A set of p53TD mutants containing putative stabilizing or destabilizing residue combinations was synthesized for a thermodynamic characterization. Unfolding experiments showed a wide range of stabilities, with T(m) values between 27 and 83 degrees C. Wild type p53TD and some highly destabilized and stabilized mutants were further characterized. Thermodynamic and biophysical data indicated that these proteins were folded tetramers, with the same overall structure, in equilibrium with unfolded monomers. An NMR study confirmed that the main structural features of p53TD are conserved in all the mutants analyzed. The thermodynamic stability of the different p53TD mutants showed a strong correlation with parameters that favor formation and stabilization of the beta-sheet. We propose that stabilization through hydrophobic interactions of key secondary structure elements might be the underlying mechanism for the strong influence of solvent-exposed residues in the stability of p53TD.

Background Intrauterine growth restriction (IUGR) is a risk factor for abnormal neurodevelopment. We studied a rabbit model of IUGR by magnetic resonance imaging (MRI) and spectroscopy (MRS), to assess in vivo brain structural and metabolic consequences, and identify potential metabolic biomarkers for clinical translation. Methods IUGR was induced in 3 pregnant rabbits at gestational day 25, by 40-50% uteroplacental vessel ligation in one horn; the contralateral horn was used as control. Fetuses were delivered at day 30 and weighted. A total of 6 controls and 5 IUGR pups underwent T2-w MRI and localized proton MRS within the first 8 hours of life, at 7T. Changes in brain tissue volumes and respective contributions to each MRS voxel were estimated by semi-automated registration of MRI images with a digital atlas of the rabbit brain. MRS data were used for: (i) absolute metabolite quantifications, using linear fitting; (ii) local temperature estimations, based on the water chemical shift; and (iii) classification, using spectral pattern analysis. Results Lower birth weight was associated with (i) smaller brain sizes, (ii) slightly lower brain temperatures, and (iii) differential metabolite profile changes in specific regions of the brain parenchyma. Specifically, we found estimated lower levels of aspartate and N-acetylaspartate (NAA) in the cerebral cortex and hippocampus (suggesting neuronal impairment), and higher glycine levels in the striatum (possible marker of brain injury). Our results also suggest that the metabolic changes in cortical regions are more prevalent than those detected in hippocampus and striatum. Conclusions IUGR was associated with brain metabolic changes in vivo, which correlate well with the neurostructural changes and neurodevelopment problems described in IUGR. Metabolic parameters could constitute non invasive biomarkers for the diagnosis and abnormal neurodevelopment of perinatal origin.

Extant disintegrins, as found in the venoms of Viperidae and Crotalidae snakes (vipers and rattlesnakes, represent a family of polypeptides that block the function of beta 1 and beta 3 integrin receptors, both potently and with a high degree of selectivity. This toxin family owes its origin to the neo-functionalization of the extracellular region of an ADAM (a disintegrin and metalloprotease) molecule recruited into the snake venom gland proteome in the Jurassic. The evolutionary structural diversification of the disintegrin scaffold, from the ancestral long disintegrins to the more recently evolved medium-sized, dimeric and short disintegrins, involved the stepwise loss of pairs of class-specific disulfide linkages and the processing of the N-terminal region. NMR and crystal structures of medium-sized, dimeric and short disintegrins have been solved. However, the structure of a long disintegrin remained unknown. The present study reports the NMR solution structures of two disulfide bond conformers of the long disintegrin bitistatin from the African puff adder Bitis arietans. The findings provide insight into how a structural domain of the extracellular region of an ADAM molecule, recruited into and selectively expressed in the snake venom gland proteome as a PIII metalloprotease in the Jurassic, has subsequently been tranformed into a family of integrin receptor antagonists.

The ATP synthase enzyme structure includes two stalk assemblies, the central stalk and the peripheral stalk. Catalysis involves rotation of the central stalk assembly together with the membrane-embedded ring of c-subunits driven by the trans-membrane proton-motive force, while the alpha and beta-subunits of F(1) are prevented from co-rotating by their attachment to the peripheral stalk. In the absence of structures of either the intact peripheral stalk or larger complexes containing it, we are studying its individual components and their interactions to build up an overall picture of its structure. Here, we describe an NMR structural characterisation of F(6), which is a 76-residue protein located in the peripheral stalk of the bovine ATP synthase and is essential for coupling between the proton-motive force and catalysis. Isolated F(6) has a highly flexible structure comprising two helices packed together through a loose hydrophobic core and connected by an unstructured linker. Analysis of chemical shifts, (15)N relaxation and RDC measurements confirm that the F(6) structure is flexible on a wide range of timescales ranging from nanoseconds to seconds. The relationship between this structure for isolated F(6) and its role in the intact peripheral stalk is discussed.

11 páginas, 4 figuras., BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases., This work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN - BIO2007-60066, SAF2008-00048, SAF30542-C01-01 and SAF2010-15512), Laboratorios SALVAT, S.A., Fundacio´n Renal Toma´s de Osma, Generalitat Valenciana Prometeo 2010/005 (partially funded with ERDF), Consolider-Ingenio 2010 (MICINN - CSD2008-00005C) and by the Generalitat Valenciana through Prometeo 2014/061. Moure was funded by a predoctoral fellowship from JAE-pre CSIC.

Bovine IF1 is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F1F0 ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF1 (44–84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

Mutants of chymotrypsin inhibitor protein 2 have previously been studied in which 4 or 10 glutamine residues were inserted into the inhibitory loop of the protein between residues 59 and 60, as potential models for the behaviour of glutamine tracts in proteins associated with polyglutamine-expansion neurodegenarative diseases. These mutants form very stable monomers, dimers and trimers. Although the cause of oligomerisation was found to be domain-swapping, it was thought that the glutamine insertions might nevertheless show evidence of weak interglutamine interactions in solution that could mimic those occurring in disease-associated proteins. In the present NMR study, we used steady-state (15)N[(1)H] NOE measurements and chemical shift comparisons to characterise the motional properties of the inserted glutamines in these CI2 mutants. We found the glutamines to be highly mobile, with no evidence of interactions amongst them in either monomers or dimers.

The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.

Neutral trans-cyanide alkenylcarbyne complexes 2a and 2b have been prepared by reaction of the complex 1a and 1b with NaCN or [Bu4N]CN. The reaction of complexes 2a and 2b with an equimolar amount of the acetonitrile complexes 1a and 1b in CH2Cl2 leads to the cationic cyanide-bridged bis(alkenylcarbyne) di-tungsten complexes 3a–d. Diisocyanide-bridged bis(alkenylcarbyne) di-tungsten complexes 4a and 4b have been synthesized by the reaction of complexes 1a and 1b with 0.5 equivalents of the diisocyanide 1,4-(CN)2C6H4. IR as well as 1H-, 31P{1H}-, 13C{1H}-, and 183W-NMR data are reported. The spectroscopic data show that in the dinuclear complexes 3a–d, the bridging CN group and the alkenylcarbyne units are located in trans positions, while in the dinuclear complexes 4a and b, the isocyanide groups of the bridging ligand 1,4-(CN)2C6H4 and the two alkenylcarbyne moieties are cis. The 183W chemical shifts of complexes 2a, 2b, 3a–d, 4a, and 4b were obtained through two-dimensional indirect 31P, 183W NMR recording techniques. A downfield shifting of 183W resonances of the cyanide-bridged dinuclear complexes 3a–d with respect to the mononuclear ones, 2a and 2b, was observed. The δ183W of isocyanide bridging dinuclear complexes 4a and 4b appear at higher field than those of the corresponding mononuclear cyanide 2a and 2b in accordance with the higher π-acceptor electron properties of the isocyanide ligand. The electrochemical behaviour of all the complexes has been investigated by cyclic voltammetry and controlled potential electrolysis in aprotic media and at a Pt (or vitreous C) electrode. Complexes 1, 2, or 3 undergo multi-electron irreversible oxidation processes involving anodically induced proton dissociation from the alkenylcarbyne ligands, and irreversible cathodic processes are also observed for all the complexes. The order of the redox potentials reflects that of the net electron π-acceptor/σ-donor character of the ligands and the ligating alkenylcarbynes are shown to behave as remarkably strong π-electron acceptors (even stronger than CO).

The reactions of [N3P3(dobp)2Cl2] and [N3P3(dobp)Cl4] with a mixture of HOC5H4N-4 and K2CO3 in acetone give the cyclotriphosphazenes [N3P3(dobp)2(OC5H4N-4)2] and [N3P3(dobp)(OC5H4N-4)4], respectively. These compounds react with [W(MeOH)(CO)5] in methanol to give mixtures of the polymetallic complexes [N3P3(dobp)2(OC5H4N-4)2{W(CO)5}x] (x = 1, 2) and [N3P3(dobp)(OC5H4N-4)4{W(CO)5}x] (x = 1–4), which are unstable in solution, slowly undergoing loss of the pentacarbonyl moiety. A complete characterization by multinuclear 1H, 15N, 31P, 183W magnetic resonance has revealed that the complexation of the N atom of one 4-oxypyridine ligand by the W(CO)5 fragment has a measurable effect on other parts of the phosphazene molecule very far away from the coordination site. The changes observed in δ183W have been used to identify the components in mixtures of compounds incorporating different numbers of tungsten atoms in the molecule. The characterization of less sensitive nuclei has been accomplished by means of indirect detection methods.

183W NMR data are reported for a series of alkenylvinylidene– and alkenylcarbyne–tungsten complexes of the type [(dppe)(CO)3WCCHR], [(dppe)(CO)3WC−CHCR2]+BF4- and [(dppe)(CO)2LWC−CHCR2] [dppe=1,2-bis(diphenylphosphino)ethane]. Spectra were recorded using inverse 2D 1H,183W- and 31P,183W{1H}-HMQC NMR spectroscopy without 183W decoupling. In some examples of the alkenylcarbyne series minor amounts of the propenylcarbyne isomer were present and the corresponding 183W chemical shifts could also be characterized. For a given complex and measuring time the proton-detected correlation spectra based on the existing long-range coupling to the metal affords a higher signal-to-noise ratio than the phosphorus-detected correlation via 1J(183W,31P). Additionally, the tilt of cross peaks in the 1H,183W HMQC spectra reveals the relative sign of the coupling of these nuclei with phosphorus. 183W chemical shift variations for compounds [(dppe)(CO)2LWC−CHCR2] (L=CO, PMe3, CH3CN or halogen) are rationalized in terms of the π-acceptor ability of the ligand. Halogen derivatives show shielding decreases in the order F

The 1H and 13C NMR spectra of 9-deoxo-9a-aza-9a-homoerythromycin A, its 11,12-hydrogenborate derivative and azithromycin 11,12-hydrogenborate were unambiguously assigned using a combination of 1D and 2D H–C correlation spectra. ©1998 John Wiley & Sons, Ltd.