Mobitz II Second-Degree Heart Block During Total Hip Arthroplasty: ACase Report.

Spontaneous intraoperative development of Mobitz II second-degree atrioventricular block is a rare event which requires decisive action on the part of anesthesiologists and anesthetists. Given that this arrhythmia can be fatal if not properly managed, it is imperative that every practitioner know how it should be managed. Currently, there is a lack of literature discussing what to expect when a patient develops this complication and what the best management strategies are. This case report describes the unexpected development of Mobitz II second-degree atrioventricular block in an elderly patient with no prior history of conduction abnormalities undergoing total hip arthroplasty and how it was managed during the perioperative period to avoid morbidity or mortality. It includes a proposed management algorithm as an easy to use guide in the management of similar clinical scenarios. While this algorithm should be familiar to anesthesiologists and experienced anesthetists, it can serve as a reference in critical situations, and may help in educating trainees.

The Pfizer Crystal Structure Database: An essential tool for structure-based design at Pfizer.

Pfizer's Crystal Structure Database (CSDB) is a key enabling technology that allows scientists on structure-based projects rapid access to Pfizer's vast library of in-house crystal structures, as well as a significant number of structures imported from the Protein Data Bank. In addition to capturing basic information such as the asymmetric unit coordinates, reflection data, and the like, CSDB employs a variety of automated methods to first ensure a standard level of annotations and error checking, and then to add significant value for design teams by processing the structures through a sequence of algorithms that prepares the structures for use in modeling. The structures are made available, both as the original asymmetric unit as submitted, as well as the final prepared structures, through REST-based web services that are consumed by several client desktop applications. The structures can be searched by keyword, sequence, submission date, ligand substructure and similarity search, and other common queries.

The Year in Cardiothoracic and Vascular Anesthesia: Selected Highlights from 2021.

This special article is the fourteenth in an annual series for the Journal of Cardiothoracic and Vascular Anesthesia. The authors thank the Editor-in-Chief, Dr. Kaplan, and the editorial board for the opportunity to continue this series; namely, the research highlights of the past year in the specialty of cardiothoracic and vascular anesthesiology. The major themes selected for 2021 are outlined in this introduction, and each highlight is reviewed in detail in the main body of the article. The literature highlights in the specialty for 2021 begin with an update on structural heart disease, with a focus on updates in arrhythmia and aortic valve disorders. The second major theme is an update on coronary artery disease, with discussion of both medical and procedural management. The third major theme is focused on the perioperative management of patients with COVID-19, with the authors highlighting literature discussing the impact of the disease on the right ventricle and thromboembolic events. The fourth and final theme is an update in heart failure, with discussion of diverse aspects of this area. The themes selected for this fourteenth special article are only a few of the diverse advances in the specialty during 2021. These highlights will inform the reader of key updates on a variety of topics, leading to improvement of perioperative outcomes for patients with cardiothoracic and vascular disease.

Delineating the Ligand-Receptor Interactions That Lead to Biased Signaling at the µ-Opioid Receptor.

Biased agonists, which selectively stimulate certain signaling pathways controlled by a G protein-coupled receptor (GPCR), hold great promise as drugs that maximize efficacy while minimizing dangerous side effects. Biased agonists of the µ-opioid receptor (µOR) are of particular interest as a means to achieve analgesia through G protein signaling without dose-limiting side effects such as respiratory depression and constipation. Rational structure-based design of biased agonists remains highly challenging, however, because the ligand-mediated interactions that are key to activation of each signaling pathway remain unclear. We identify several compounds for which the R- and S-enantiomers have distinct bias profiles at the µOR. These compounds serve as excellent comparative tools to study bias because the identical physicochemical properties of enantiomer pairs ensure that differences in bias profiles are due to differences in interactions with the µOR binding pocket. Atomic-level simulations of compounds at µOR indicate that R- and S-enantiomers adopt different poses that form distinct interactions with the binding pocket. A handful of specific interactions with highly conserved binding pocket residues appear to be responsible for substantial differences in arrestin recruitment between enantiomers. Our results offer guidance for rational design of biased agonists at µOR and possibly at related GPCRs.

Managing COVID-19 from the epicenter: adaptations and suggestions based on experience.

In March 2020, the New York City metropolitan area became the epicenter of the United States' SARS-CoV-2 pandemic and the surge of new cases threatened to overwhelm the area's hospital systems. This article describes how an anesthesiology department at a large urban academic hospital rapidly adapted and deployed to meet the threat head-on. Topics included are preparatory efforts, development of a team-based staffing model, and a new strategy for resource management. While still maintaining a fully functioning operating theater, discrete teams were deployed to both COVID-19 and non-COVID-19 intensive care units, rapid response/airway management team, the difficult airway response team, and labor and delivery. Additional topics include the creation of a temporary 'pop-up' anesthesiology-run COVID-19 intensive care unit utilizing anesthesia machines for monitoring and ventilatory support as well as the development of a simulation and innovation team that was instrumental in the rapid prototyping of a controlled split-ventilation system and conversion of readily available BIPAP units into emergency ventilators. As the course of the disease is uncertain, the goal of this article is to assist others in preparation for what may come next with COVID-19 as well as potential future pandemics.

Design and Identification of a Novel, Functionally Subtype Selective GABAA Positive Allosteric Modulator (PF-06372865).

The design, optimization, and evaluation of a series of novel imidazopyridazine-based subtype-selective positive allosteric modulators (PAMs) for the GABAA ligand-gated ion channel are described. From a set of initial hits multiple subseries were designed and evaluated based on binding affinity and functional activity. As designing in the desired level of functional selectivity proved difficult, a probability-based assessment was performed to focus the project's efforts on a single subseries that had the greatest odds of delivering the target profile. These efforts ultimately led to the identification of two precandidates from this subseries, which were advanced to preclinical safety studies and subsequently to the identification of the clinical candidate PF-06372865.

Pharmacology in translation: the preclinical and early clinical profile of the novel α2/3 functionally selective GABAA receptor positive allosteric modulator PF-06372865.

BACKGROUND AND PURPOSE: Benzodiazepines, non-selective positive allosteric modulators (PAMs) of GABAA receptors, have significant side effects that limit their clinical utility. As many of these side effects are mediated by the α1 subunit, there has been a concerted effort to develop α2/3 subtype-selective PAMs. EXPERIMENTAL APPROACH: In vitro screening assays were used to identify molecules with functional selectivity for receptors containing α2/3 subunits over those containing α1 subunits. In vivo receptor occupancy (RO) was conducted, prior to confirmation of in vivo α2/3 and α1 pharmacology through quantitative EEG (qEEG) beta frequency and zolpidem drug discrimination in rats respectively. PF-06372865 was then progressed to Phase 1 clinical trials. KEY RESULTS: PF-06372865 exhibited functional selectivity for those receptors containing α2/3/5 subunits, with significant positive allosteric modulation (90-140%) but negligible activity (≤20%) at GABAA receptors containing α1 subunits. PF-06372865 exhibited concentration-dependent occupancy of GABAA receptors in preclinical species. There was an occupancy-dependent increase in qEEG beta frequency and no generalization to a GABAA α1 cue in the drug-discrimination assay, clearly demonstrating the lack of modulation at the GABAA receptors containing an α1 subtype. In a Phase 1 single ascending dose study in healthy volunteers, evaluation of the pharmacodynamics of PF-06372865 demonstrated a robust increase in saccadic peak velocity (a marker of α2/3 pharmacology), increases in beta frequency qEEG and a slight saturating increase in body sway. CONCLUSIONS AND IMPLICATIONS: PF-06372865 has a unique clinical pharmacology profile and a highly predictive translational data package from preclinical species to the clinical setting.

Analysis of Differential Efficacy and Affinity of GABAA (α1/α2) Selective Modulators.

Selective modulators of the γ-amino butyric acid (GABAA) family of receptors have the potential to treat a range of disease states related to cognition, pain, and anxiety. While the development of various α subunit-selective modulators is currently underway for the treatment of anxiety disorders, a mechanistic understanding of the correlation between their bioactivity and efficacy, based on ligand-target interactions, is currently still lacking. In order to alleviate this situation, in the current study we have analyzed, using ligand- and structure-based methods, a data set of 5440 GABAA modulators. The Spearman correlation (ρ) between binding activity and efficacy of compounds was calculated to be 0.008 and 0.31 against the α1 and α2 subunits of GABA receptor, respectively; in other words, the compounds had little diversity in structure and bioactivity, but they differed significantly in efficacy. Two compounds were selected as a case study for detailed interaction analysis due to the small difference in their structures and affinities (ΔpKi(comp1_α1 - comp2_α1) = 0.45 log units, ΔpKi(comp1_α2 - comp2_α2) = 0 log units) as compared to larger relative efficacies (ΔRE(comp1_α1 - comp2_α1) = 1.03, ΔRE(comp1_α2 - comp2_α2) = 0.21). Docking analysis suggested that His-101 is involved in a characteristic interaction of the α1 receptor with both compounds 1 and 2. Residues such as Phe-77, Thr-142, Asn-60, and Arg-144 of the γ chain of the α1γ2 complex also showed interactions with heterocyclic rings of both compounds 1 and 2, but these interactions were disturbed in the case of α2γ2 complex docking results. Binding pocket stability analysis based on molecular dynamics identified three substitutions in the loop C region of the α2 subunit, namely, G200E, I201T, and V202I, causing a reduction in the flexibility of α2 compared to α1. These amino acids in α2, as compared to α1, were also observed to decrease the vibrational and dihedral entropy and to increase the hydrogen bond content in α2 in the apo state. However, freezing of both α1 and α2 was observed in the ligand-bound state, with an increased number of internal hydrogen bonds and increased entropy. Therefore, we hypothesize that the amino acid differences in the loop C region of α2 are responsible for conformational changes in the protein structure compared to α1, as well as for the binding modes of compounds and hence their functional signaling.

Targeting the cAMP and Transforming Growth Factor-ß Pathway Increases Proliferation to Promote Re-Epithelialization of Human Stem Cell-Derived Retinal Pigment Epithelium.

UNLABELLED: Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell-derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESCs) differentiated into RPE progeny have the potential to provide an unlimited supply of cells for transplantation, but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield after differentiation. We show that RPE epithelialization is a density-dependent process, and cells seeded at low density fail to epithelialize. We demonstrate that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of transforming growth factor-ß (TGF-ß) signaling. This results in enhanced uptake of epithelial identity, even in cultures seeded at low density. In line with these findings, targeted manipulation of the TGF-ß pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cell-derived RPE. Modulation of these pathways has the potential to favorably impact scalability and clinical translation of hESC-derived RPE as a cell therapy. SIGNIFICANCE: Stem cell-derived retinal pigment epithelium (RPE) is currently being evaluated as a cell-replacement therapy for macular degeneration. This work shows that the process of generating RPE in vitro is regulated by the cAMP and transforming growth factor-ß signaling pathway. Modulation of these pathways by small molecules, as identified by phenotypic screening, leads to an increased efficiency of generating RPE cells with a higher yield. This can have a potential impact on manufacturing transplantation-ready cells at large scale and is advantageous for clinical studies using this approach in the future.

Voltage gated sodium channels as drug discovery targets.

Voltage-gated sodium (NaV) channels are a family of transmembrane ion channel proteins. They function by forming a gated, water-filled pore to help establish and control cell membrane potential via control of the flow of ions between the intracellular and the extracellular environments. Blockade of NaVs has been successfully accomplished in the clinic to enable control of pathological firing patterns that occur in a diverse range of conditions such as chronic pain, epilepsy, and cardiac arrhythmias. First generation sodium channel modulator drugs, despite low inherent subtype selectivity, preferentially act on over-excited cells which reduces undesirable side effects in the clinic. However, the limited therapeutic indices observed with the first generation demanded a new generation of sodium channel inhibitors. The structure, function and the state of the art in sodium channel modulator drug discovery are discussed in this chapter.

An analysis of the attrition of drug candidates from four major pharmaceutical companies.

The pharmaceutical industry remains under huge pressure to address the high attrition rates in drug development. Attempts to reduce the number of efficacy- and safety-related failures by analysing possible links to the physicochemical properties of small-molecule drug candidates have been inconclusive because of the limited size of data sets from individual companies. Here, we describe the compilation and analysis of combined data on the attrition of drug candidates from AstraZeneca, Eli Lilly and Company, GlaxoSmithKline and Pfizer. The analysis reaffirms that control of physicochemical properties during compound optimization is beneficial in identifying compounds of candidate drug quality and indicates for the first time a link between the physicochemical properties of compounds and clinical failure due to safety issues. The results also suggest that further control of physicochemical properties is unlikely to have a significant effect on attrition rates and that additional work is required to address safety-related failures. Further cross-company collaborations will be crucial to future progress in this area.

Discovery of a Selective TRPM8 Antagonist with Clinical Efficacy in Cold-Related Pain.

The transient receptor potential (TRP) family of ion channels comprises nonselective cation channels that respond to a wide range of chemical and thermal stimuli. TRPM8, a member of the melastatin subfamily, is activated by cold temperatures (<28 °C), and antagonists of this channel have the potential to treat cold induced allodynia and hyperalgesia. However, TRPM8 has also been implicated in mammalian thermoregulation and antagonists have the potential to induce hypothermia in patients. We report herein the identification and optimization of a series of TRPM8 antagonists that ultimately led to the discovery of PF-05105679. The clinical finding with this compound will be discussed, including both efficacy and its ability to affect thermoregulation processes in humans.

Ion channels as therapeutic targets: a drug discovery perspective.

Ion channels are membrane proteins expressed in almost all living cells. The sequencing of the human genome has identified more than 400 putative ion channels, but only a fraction of these have been cloned and functionally tested. The widespread tissue distribution of ion channels, coupled with the plethora of physiological consequences of their opening and closing, makes ion-channel-targeted drug discovery highly compelling. However, despite some important drugs in clinical use today, as a class, ion channels remain underexploited in drug discovery and many existing drugs are poorly selective with significant toxicities or suboptimal efficacy. This Perspective seeks to review the ion channel family, its structural and functional features, and the diseases that are known to be modulated by members of the family. In particular, we will explore the structure and properties of known ligands and consider the future prospects for drug discovery in this challenging but high potential area.

5-(2-Pyrimidinyl)-imidazo[1,2-a]pyridines are antibacterial agents targeting the ATPase domains of DNA gyrase and topoisomerase IV.

Dual inhibitors of bacterial gyrB and parE based on a 5-(2-pyrimidinyl)-imidazo[1,2-a]pyridine template exhibited MICs (microg/mL) of 0.06-64 (Sau), 0.25-64 (MRSA), 0.06-64 (Spy), 0.06-64 (Spn), and 0.03-64 (FQR Spn). Selected examples were efficacious in mouse sepsis and lung infection models at <50mg/kg (PO dosing).

Selective tumor cell targeting using low-affinity, multivalent interactions.

This report highlights the advantages of low-affinity, multivalent interactions to recognize one cell type over another. Our goal was to devise a strategy to mediate selective killing of tumor cells, which are often distinguished from normal cells by their higher levels of particular cell surface receptors. To test whether multivalent interactions could lead to highly specific cell targeting, we used a chemically synthesized small-molecule ligand composed of two distinct motifs: (1) an Arg-Gly-Asp (RGD) peptidomimetic that binds tightly (Kd approximately 10(-9)M) to alphavbeta3 integrins and (2) the galactosyl-alpha(1-3)galactose (alpha-Gal epitope), which is recognized by human anti-alpha-galactosyl antibodies (anti-Gal). Importantly, anti-Gal binding requires a multivalent presentation of carbohydrate residues; anti-Gal antibodies interact weakly with the monovalent oligosaccharide (Kd approximately 10(-5)M) but bind tightly (Kd approximately 10(-11) M) to multivalent displays of alpha-Gal epitopes. Such a display is generated when the bifunctional conjugate decorates a cell possessing a high level of alphavbeta3 integrin; the resulting cell surface, which presents many alpha-Gal epitopes, can recruit anti-Gal, thereby triggering complement-mediated lysis. Only those cells with high levels of the integrin receptor are killed. In contrast, doxorubicin tethered to the RGD-based ligand affords indiscriminate cell death. These results highlight the advantages of exploiting the type of the multivalent recognition processes used by physiological systems to discriminate between cells. The selectivity of this strategy is superior to traditional, abiotic, high-affinity targeting methods. Our results have implications for the treatment of cancer and other diseases characterized by the presence of deleterious cells.

Bifunctional ligands that target cells displaying the alpha v beta3 integrin.

Strategies to eliminate tumor cells have long been sought. We envisioned that a small molecule could be used to decorate the offending cells with immunogenic carbohydrates and evoke an immune response. To this end, we describe the synthesis of bifunctional ligands possessing two functional motifs: one binds a cell-surface protein and the other binds a naturally occurring human antibody. Our conjugates combine an RGD-based peptidomimetic, to target cells displaying the alpha v beta3 integrin, with the carbohydrate antigen galactosyl-alpha(1-3)galactose [Galalpha(1-3)Gal or alpha-Gal]. To generate such bifunctional ligands, we designed and synthesized RGD mimetics 1 b and 2 c, which possess a free amino group for modification. These compounds were used to generate bifunctional derivatives 1 c and 2 d, with dimethyl squarate serving as the linchpin; thus, our synthetic approach is modular. To evaluate the binding of our peptidomimetics to the target alpha v beta3-displaying cells, we implemented a cell-adhesion assay. Results from this assay indicate that the designed, small-molecule ligands inhibit alpha v beta3-dependent cell adhesion. Additionally, our most effective bifunctional ligand exhibits a high degree of selectivity (4000-fold) for alpha v beta3 over the related alpha v beta5 integrin, a result that augurs its utility in specific cell targeting. Finally, we demonstrate that the bifunctional ligands can bind to alpha v beta3-positive cells and recruit human anti-Gal antibodies. These results indicate that both the integrin-binding and the anti-Gal-binding moieties can act simultaneously. Bifunctional conjugates of this type can facilitate the development of new methods for targeting cancer cells by exploiting endogenous antibodies. We anticipate that our modifiable alpha v beta3-binding ligands will be valuable in a variety of applications, including drug delivery and tumor targeting.

Stereoselective synthesis of the C(1)-C(11) fragment of peloruside A.

A highly stereoselective synthesis of the C(1)-C(11) fragment 4 of peloruside A has been accomplished via a stereoselective double allylboration and an intramolecular epoxide opening to provide the functionally dense C(3)-C(11) segment 14. A glycolate aldol reaction was then employed to introduce the remaining stereocenters at C(2)-C(3). [reaction: see text]

Conserved amplification of chemotactic responses through chemoreceptor interactions.

Many bacteria concentrate their chemoreceptors at the cell poles. Chemoreceptor location is important in Escherichia coli, since chemosensory responses are sensitive to receptor proximity. It is not known, however, whether chemotaxis in other bacteria is similarly regulated. To investigate the importance of receptor-receptor interactions in other bacterial species, we synthesized saccharide-bearing multivalent ligands that are designed to cluster relevant chemoreceptors. As has been shown with E. coli, we demonstrate that the behaviors of Bacillus subtilis, Spirochaete aurantia, and Vibrio furnissii are sensitive to the valence of the chemoattractant. Moreover, in B. subtilis, chemotactic responses to serine were increased by pretreatment with saccharide-bearing multivalent ligands. This result indicates that, as in E. coli, signaling information is transferred among chemoreceptors in B. subtilis. These results suggest that interreceptor communication may be a general mechanism for modulating chemotactic responses in bacteria.

Synthesis and applications of end-labeled neoglycopolymers.

[reaction: see text] Neoglycopolymers that vary in length and contain a single fluorescent reporter group were synthesized using ring-opening metathesis polymerization (ROMP). The utility of these materials is demonstrated by the development of a cellular binding assay for L-selectin, a cell surface protein that plays a role in inflammation. The data reveal that these multivalent ligands interact with multiple copies of L-selectin.

Selective immobilization of multivalent ligands for surface plasmon resonance and fluorescence microscopy.

Cell surface multivalent ligands, such as proteoglycans and mucins, are often tethered by a single attachment point. In vitro, however, it is difficult to immobilize multivalent ligands at single sites due to their heterogeneity. Moreover, multivalent ligands often lack a single group with reactivity orthogonal to other functionality in the ligand. Biophysical analyses of multivalent ligand-receptor interactions would benefit from the availability of strategies for uniform immobilization of multivalent ligands. To this end, we report the design and synthesis of a multivalent ligand that has a single terminal orthogonal functional group and we demonstrate that this material can be selectively immobilized onto a surface suitable for surface plasmon resonance (SPR) experiments. The polymeric ligand we generated displays multiple copies of 3,6-disulfogalactose, and it can bind to the cell adhesion molecules P- and L-selectin. Using SPR measurements, we found that surfaces displaying our multivalent ligands bind specifically to P- and L-selectin. The affinities of P- and L-selectin for surfaces displaying the multivalent ligand are five- to sixfold better than the affinities for a surface modified with the corresponding monovalent ligand. In addition to binding soluble proteins, surfaces bearing immobilized polymers bound to cells displaying L-selectin. Cell binding was confirmed by visualizing adherent cells by fluorescence microscopy. Together, our results indicate that synthetic surfaces can be created by selective immobilization of multivalent ligands and that these surfaces are capable of binding soluble and cell-surface-associated receptors with high affinity.