Conformational changes in the G protein Gs induced by the ß2 adrenergic receptor.

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human ß(2) adrenergic receptor (ß(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the ß(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the ß(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the ß-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and ß-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

Crystal structure of the ß2 adrenergic receptor-Gs protein complex.

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ß(2) adrenergic receptor (ß(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ß(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ß(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ß(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

Hydroxylation and N-dechloroethylation of Ifosfamide and deuterated Ifosfamide by the human cytochrome p450s and their commonly occurring polymorphisms.

The hydroxylation and N-dechloroethylation of deuterated ifosfamide (d4IFO) and ifosfamide (IFO) by several human P450s have been determined and compared. d4IFO was synthesized with deuterium at the alpha and alpha' carbons to decrease the rate of N-dechloroethylation and thereby enhance hydroxylation of the drug at the 4' position. The purpose was to decrease the toxic and increase the efficacious metabolites of IFO. For all of the P450s tested, hydroxylation of d4IFO was improved and dechloroethylation was reduced as compared with nondeuterated IFO. Although the differences were not statistically significant, the trend favoring the 4'-hydroxylation pathway was noteworthy. CYP3A5 and CYP2C19 were the most efficient enzymes for catalyzing IFO hydroxylation. The importance of these enzymes in IFO metabolism has not been reported previously and warrants further investigation. The catalytic ability of the common polymorphisms of CYP2B6 and CYP2C9 for both reactions were tested with IFO and d4IFO. It was determined that the commonly expressed polymorphisms CYP2B6*4 and CYP2B6*6 had reduced catalytic ability for IFO compared with CYP2B6*1, whereas CYP2B6*7 and CYP2B6*9 had enhanced catalytic ability. As with the wild-type enzymes, d4IFO was more readily hydroxylated by the polymorphic variants than IFO, and d4IFO was not dechloroethylated by any of the polymorphic forms. We also assessed the use of specific inhibitors of P450 to favor hydroxylation in human liver microsomes. We were unable to separate the pathways with these experiments, suggesting that multiple P450s are responsible for catalyzing both metabolic pathways for IFO, which is not observed with the closely related drug cyclophosphamide.

Pharmacogenetic considerations in the elderly patient.

OBJECTIVE: To outline how the inclusion of pharmacogenetic data lends additional information in the overall decision making relative to drug therapy in the elderly patient. DATA SOURCES: The National Center for Biotechnology's PubMed database was searched for relevant pharmacogenetic-based dosing guidelines, as well as papers discussing drug use, and pharmacogenetics in the elderly. Google Scholar was also searched for the related documents. STUDY SELECTION: Papers cited were those that presented a rationale for drug therapy in the elderly, presented pharmacogenetic-based dosing guidelines with supporting information, and specifically discussed pharmacogenetics and other therapeutic principles relative to drug therapy in the elderly. DATA SYNTHESIS: Specific examples were extracted for presentation where data on drug use in the elderly corresponded with pharmacogenetic information. Specific examples were selected to illustrate pharmacogenetic influences on medications of clinical significance in the elderly population including meperidine, tramadol, amitriptyline, nortriptyline, flecainide, and propafenone. These medications were identified as intersecting points in the Beers criteria and pharmacogenetic guidelines provided by the Clinical Pharmacogenetics Implementation Consortium and the Dutch Pharmacogenetics Working Group, or where mechanisms of pharmacogenetic influences were applicable. CONCLUSIONS: Inclusion of pharmacogenetic data/information in the decision-making process may help the clinician to more appropriately guide therapy in the elderly patient.

Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex.

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied ß(2)-adrenoceptor (ß(2)AR) in complex with the heterotrimeric G protein Gs (Gαsßγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

The effect of ritonavir on human CYP2B6 catalytic activity: heme modification contributes to the mechanism-based inactivation of CYP2B6 and CYP3A4 by ritonavir.

The mechanism-based inactivation of human CYP2B6 by ritonavir (RTV) in a reconstituted system was investigated. The inactivation is time, concentration, and NADPH dependent and exhibits a K(I) of 0.9 µM, a k(inact) of 0.05 min⁻¹, and a partition ratio of approximately 3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the protonated molecular ion of RTV exhibits an m/z at 721 and its two major metabolites are an oxidation product with MH⁺ at m/z 737 and a deacylated product with MH⁺ at m/z 580. Inactivation of CYP2B6 by incubation with 10 µM RTV for 10 min resulted in an approximately 50% loss of catalytic activity and native heme, but no modification of the apoprotein was observed. RTV was found to be a potent mixed-type reversible inhibitor (K(i) = 0.33 µM) and a type II ligand (spectral dissociation constant-K(s) = 0.85 µM) of CYP2B6. Although previous studies have demonstrated that RTV is a potent mechanism-based inactivator of CYP3A4, the molecular mechanism responsible for the inactivation has not been determined. Here, we provide evidence that RTV inactivation of CYP3A4 is due to heme destruction with the formation of a heme-protein adduct. Similar to CYP2B6, there is no significant modification of the apoprotein. Furthermore, LC-MS/MS analysis revealed that both CYP3A4 and human liver microsomes form an RTV-glutathione conjugate having a MH⁺ at m/z 858 during metabolism of RTV, suggesting the formation of an isocyanate intermediate leading to formation of the conjugate.

A Genetics-Focused Lens on Social Constructs in Pharmacy Education.

OBJECTIVE: Incorporating diversity, equity, inclusion, and anti-racism principles into clinical and didactic education is essential because each influence cognitive and affective attitudes in pharmacy practice. Educators must learn from the past to enlighten the future. For example, race is a social construct, not a biological construct. However, it persistently acts as a surrogate for determining medical diagnoses and treatment. FINDINGS: Precision medicine and pharmacogenomics can serve as a basis for deconstructing social constructs surrounding race and other social determinants of health. SUMMARY: In this review, the authors highlight why using race in health education will lead to less-than-optimal clinical decisions and discuss best practices for incorporating diversity, equity, inclusion, and anti-racism into health education from a pharmacogenomic-based perspective.

An interprofessional education experience to promote the role of the pharmacist in precision medicine.

BACKGROUND: An interprofessional (IP) experience was created that demonstrated the roles and responsibilities of pharmacists and physicians in clinical implementation of pharmacogenomics (PGx). The IP experience focused on PGx-themed patient cases and application of genotyping results to drug therapy management. INTERPROFESSIONAL EDUCATION ACTIVITY: In 2016 and 2017, third-year pharmacy students and first-year medical students were placed on interprofessional teams with two to three students each. The teams resolved PGx patient cases, medical students wrote prescriptions for altered drug therapy based on the PGx profiles of the patients, and pharmacy students assessed and provided feedback to medical students about the prescriptions. Student could also volunteer to be genotyped for CYP2C19*2, and the results were compared. DISCUSSION: The IP experience significantly enhanced PGx knowledge and increased the confidence of using PGx in patient cases for the majority of participants. The experience did not increase the recognition of each discipline's role in precision medicine in a statistically significant manner. Accurate prescription writing was challenging for the first-year medical students (44.3% prescriptions written correctly). The genotyping results did not deviate from a Hardy Weinberg equilibrium for this population. IMPLICATIONS: IP experiences focused on PGx present an ideal opportunity to educate and initiate collaborations between pharmacists and physicians and to promote utilization of PGx in precision medicine. The roles and responsibilities for each discipline can be easily recreated in an IP experience to provide robust training to the students.

Complex patient cases solved by near-peer integrated teams provides leadership, professionalism, and peer-teaching opportunities.

BACKGROUND AND PURPOSE: Near-peer teaching is commonly used in experiential learning, residency training, and teaching certificate programs, but data on near-peer teaching in didactic course work are lacking. An innovative, near-peer learning opportunity that encouraged leadership and professionalism is described. EDUCATIONAL ACTIVITY AND SETTING: Thirty-six teams comprised of first-, second-, and third-year pharmacy students presented a 15-min oncology patient case to faculty reviewers. Presentations were focused on calculations, medicinal chemistry, pharmacology, and therapeutic application. Students were evaluated on communication skills, presentation skills, and presentation content. Additionally, students provided feedback about their perceptions of the laboratory experience. A chi-square test was used to determine significance or parity in student responses. FINDINGS: The majority of the students (82%) felt the teams worked well together and 85% indicated that they played an important role on their team, with second- and third-year students rating this item significantly higher than first year students. Additionally, 85% of participating students expressed that this experience provided an opportunity to demonstrate professionalism. SUMMARY: Integration of first-, second-, and third-year pharmacy students into teams was an innovative and effective method to provide opportunity for leadership, professionalism, and near-peer teaching through evaluation and presentation of an oncology patient case.

A review of the influence of functional group modifications to the core scaffold of synthetic cathinones on drug pharmacokinetics.

RATIONALE: The synthetic cathinones are a class of designer drugs of abuse that share a common core scaffold. The pharmacokinetic profiles of the synthetic cathinones vary based on the substitutions to the core scaffold. OBJECTIVES: To provide a summary of the literature regarding the pharmacokinetic characteristics of the synthetic cathinones, with a focus on the impact of the structural modifications to the pharmacokinetics. RESULTS: In many, but not all, instances the pharmacokinetic characteristics of the synthetic cathinones can be reasonably predicted based on the substitutions to the core scaffold. Mephedrone and methylone are chemically alike and have similar Tmax and t1/2 in male rats. MDPV, a structurally distinct synthetic cathinone from mephedrone and methylone, has a lower Tmax and t1/2. Increasing the length of the alkyl chain on the α position of methylone, to produce pentylone, results in increased plasma concentrations and longer t1/2. Metabolism of the synthetic cathinones is reasonably predictable based on the chemical structure, and several phase I metabolites retain pharmacodynamic activity. CYP2D6 is implicated in the metabolism of all of the synthetic cathinones, and other P450s (CYP1A2, CYP2B6, and CYP2C19) are known to contribute variably to the metabolism of specific synthetic cathinones. CONCLUSIONS: Continued research will lead to a better understanding of the pharmacokinetic changes associated with structural modifications to the cathinone scaffold, and potentially in the long range, enhanced overdose and addiction therapy. Additionally, the areas of polydrug use and pharmacogenetics have been largely overlooked with regard to synthetic cathinones.

Food restriction alters N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride (pramipexole)-induced yawning, hypothermia, and locomotor activity in rats: evidence for sensitization of dopamine D2 receptor-mediated effects.

Food restriction enhances sensitivity to the reinforcing effects of a variety of drugs of abuse including opiates, nicotine, and psychostimulants. Food restriction has also been shown to alter a variety of behavioral and pharmacological responses to dopaminergic agonists, including an increased sensitivity to the locomotor stimulatory effects of direct- and indirect-dopamine agonists, elevated extracellular dopamine levels in responses to psychostimulants, as well as suppression of agonist-induced yawning. Behavioral and molecular studies suggest that augmented dopaminergic responses observed in food-restricted animals result from a sensitization of the dopamine D2 receptor; however, little is known about how food restriction affects dopamine D3 receptor function. The current studies were aimed at better defining the effects of food restriction on D2 and D3 receptor function by assessing the capacity of N'-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine dihydrochloride (pramipexole) to induce yawning, penile erection (PE), hypothermia, and locomotor activity in free-fed and food-restricted rats. Food restriction resulted in a suppression of pramipexole-induced yawning, a sensitized hypothermic response, and an enhanced locomotor response to pramipexole, effects that are suggestive of an enhanced D2 receptor activity; no effect on pramipexole-induced PE was observed. Antagonist studies further supported a food restriction-induced enhancement of the D2 receptor activity because the D2 antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) recovered pramipexole-induced yawning to free-fed levels, whereas yawning and PE were suppressed following pretreatment with the D3 antagonist N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide hydrochloride (PG01037). The results of the current studies suggest that food restriction sensitized rats to the D2-mediated effects of pramipexole while having no effect on the D3-mediated effects of pramipexole.

P&T and Me.

Very few occasions bring more stress to a faculty member than the promotion and/or tenure (P&T) process. In this commentary, two recent chairs of P&T committees give their advice to future petitioners. Subtopics of the editorial discuss knowing the expectations, finding mentors to guide you, understanding the role of peer reviewers and preparing your dossier.

Cancer cell sensitivity to bortezomib is associated with survivin expression and p53 status but not cancer cell types.

BACKGROUND: Survivin is known playing a role in drug resistance. However, its role in bortezomib-mediated inhibition of growth and induction of apoptosis is unclear. There are conflicting reports for the effect of bortezomib on survivin expression, which lacks of a plausible explanation. METHODS: In this study, we tested cancer cells with both p53 wild type and mutant/null background for the relationship of bortezomib resistance with survivin expression and p53 status using MTT assay, flow cytometry, DNA fragmentation, caspase activation, western blots and RNAi technology. RESULTS: We found that cancer cells with wild type p53 show a low level expression of survivin and are sensitive to treatment with bortezomib, while cancer cells with a mutant or null p53 show a high level expression of survivin and are resistant to bortezomib-mediated apoptosis induction. However, silencing of survivin expression utilizing survivin mRNA-specific siRNA/shRNA in p53 mutant or null cells sensitized cancer cells to bortezomib mediated apoptosis induction, suggesting a role for survivin in bortezomib resistance. We further noted that modulation of survivin expression by bortezomib is dependent on p53 status but independent of cancer cell types. In cancer cells with mutated p53 or p53 null, bortezomib appears to induce survivin expression, while in cancer cells with wild type p53, bortezomib downregulates or shows no significant effect on survivin expression, which is dependent on the drug concentration, cell line and exposure time. CONCLUSIONS: Our findings, for the first time, unify the current inconsistent findings for bortezomib treatment and survivin expression, and linked the effect of bortezomib on survivin expression, apoptosis induction and bortezomib resistance in the relationship with p53 status, which is independent of cancer cell types. Further mechanistic studies along with this line may impact the optimal clinical application of bortezomib in solid cancer therapeutics.