Computational Investigations of the Lithium Superoxide Dimer Rearrangement on Noisy Quantum Devices.

Quantum chemistry studies of biradical systems are challenging due to the required multiconfigurational nature of the wavefunction. In this work, Variational Quantum Eigensolver (VQE) is used to compute the energy profile for the lithium superoxide dimer rearrangement, involving biradical species, on quantum simulators and devices. Considering that current quantum devices can only handle limited number of qubits, we present guidelines for selecting an appropriate active space to perform computations on chemical systems that require many qubits. We show that with VQE performed with a quantum simulator reproduces results obtained with full-configuration interaction (Full CI) for the chosen active space. However, results deviate from exact values by about 39 mHa for calculations on a quantum device. This deviation can be improved to about 4 mHa using the readout mitigation approach and can be further improved to 2 mHa, approaching chemical accuracy, using the state tomography technique to purify the calculated quantum state.

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis.

The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the ß-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.

Interaction of serum amyloid P component with hexanoyl bis(D-proline) (CPHPC).

Under physiological conditions, the pentameric human plasma protein serum amyloid P component (SAP) binds hexanoyl bis(D-proline) (R-1-{6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl}pyrrolidine-2-carboxylic acid; CPHPC) through its D-proline head groups in a calcium-dependent interaction. Cooperative effects in binding lead to a substantial enhancement of affinity. Five molecules of the bivalent ligand cross-link and stabilize pairs of SAP molecules, forming a decameric complex that is rapidly cleared from the circulation by the liver. Here, it is reported that X-ray analysis of the SAP complex with CPHPC and cadmium ions provides higher resolution detail of the interaction than is observed with calcium ions. Conformational isomers of CPHPC observed in solution by HPLC and by X-ray analysis are compared with the protein-bound form. These are discussed in relation to the development of CPHPC to provide SAP depletion for the treatment of amyloidosis and other indications.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Targeting C-reactive protein for the treatment of cardiovascular disease.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Molecular dissection of Alzheimer's disease neuropathology by depletion of serum amyloid P component.

New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Abeta amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated tau protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.

Morphological differences between ß(2) -microglobulin in fibrils and inclusion bodies.

Over expression of proteins in E. coli frequently results in the production of inclusion bodies. Although ß(2) -microglobulin frequently forms fibrillar structures, our studies reveal significant differences between the protein in fibrils and inclusion bodies. This suggests that the formation of fibrils in inclusion bodies is dependent on the propensity of the protein to form fibrillar structures.

Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component.

The normal physiological roles of the phylogenetically conserved human plasma proteins C-reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities are in clinical development as both proteins have significant pathophysiological effects, SAP in promoting amyloidosis and CRP in exacerbating ischemic injury. Both proteins bind to phosphoethanolamine and we show here that, under physiological conditions, phosphoethanolamine is bound with higher affinity by human SAP than by human CRP. An explanation is provided by X-ray crystal structures that show SAP residue Tyr74 allowing additional hydrophobic protein-ligand interactions compared with the equivalent Thr76 of CRP. Docking simulations show many more low energy positions for phosphoethanolamine bound by CRP than by SAP and are consistent with the crystallographic and functional binding results. These fundamental observations on structure-activity relationships will aid the design of improved pentraxin targeting drugs.

Structure of human porphobilinogen deaminase at 2.8 A: the molecular basis of acute intermittent porphyria.

Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.

Trafficked.

This first-person narrative describes some of the barriers to caring well for patients at the intersection of human trafficking and substance use disorder. I canvass some of the ethical considerations regarding these patients' autonomy and call for establishing and using evidence-based practice to manage these complex scenarios.

Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants.

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.

Bifunctional crosslinking ligands for transthyretin.

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR-ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.

Contrast-induced nephropathy in critical care.

Contrast-induced nephropathy is an iatrogenic disease caused by the administration of iodinated contrast material to certain at-risk patients. The clinical features include renal failure, with oliguria, anuria, and electrolyte derangements. Contrast-induced nephropathy can prolong hospitalization, result in greater morbidity and mortality, and increase patients' costs. A variety of preventive and treatment strategies exist, including use of alternative imaging. Critical care nurses need to understand the nephropathy and the patients at risk and to develop a familiarity with prevention, treatment, and outcome.

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors.

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.