A Learning Health System Approach to Increasing Human Papillomavirus Immunizations Among Young Adults.

INTRODUCTION Immunization rates against the human papillomavirus (HPV) remain suboptimal in the young adult population. Little is known about the most effective means for encouraging vaccination in this population. METHODS The authors conducted a clinical trial of 3 methods to encourage HPV vaccination in a large Northern California integrated Health Plan. Young adults aged 18-26 with evidence of insufficient HPV vaccination were sent a bulk secure message from the Health Plan (standard outreach); those who did not respond were randomized to no further outreach, a second, personalized secure message from a specific practitioner, or a letter mailed to their home. The primary outcome was receipt of at least 1 HPV vaccine within 3 months following the initial bulk secure message. RESULTS In total, 7718 young adults were randomized. After 3 months, 86 patients (3.5%) who received no additional outreach obtained an immunization, compared with 114 (4.6%) who received the second secure message (p = 0.05) and 126 (5.1%) who received the mailed letter (p = 0.006). DISCUSSION Supplemental mailed or personalized electronic messages increased vaccination beyond no additional intervention, although gains were not clinically meaningful. These findings highlight the need for more successful alternatives to encourage uptake of such preventive health interventions among young adults. The successful conduct of this rapid-cycle, randomized trial showed that such evaluations are feasible, providing actionable data to inform implementation strategies. CONCLUSIONS Further study is needed to identify effective strategies for improving preventive health uptake in this important and underserved population. Rapid-cycle randomized evaluation strategies can provide critical information to focus efforts for achieving this goal.

Incidental paratracheal lymph node lung adenocarcinoma in a patient with goiter: a case report.

Background: Paratracheal lymph nodes can be sites of metastasis for a variety of malignancies, but these metastases are treated differently depending on the tissue of origin. We describe a patient who underwent combined thoracoscopic and cervical resection of a multinodular goiter who was found to have incidental lung adenocarcinoma in an adjacent paratracheal lymph node despite having no pulmonary nodules. Case Description: A 62-year-old male with longstanding substernal multinodular goiter presented to his primary care doctor with continued growth of his goiter. After repeatedly declining surgery, he became amenable to resection and underwent right video-assisted thoracoscopic and cervical approaches. An incidentally found separate large right paratracheal lymph node was also discovered and completely resected. Final pathology of the thyroid mass revealed hyperplastic thyroid nodules consistent with a benign goiter. However, the separate right paratracheal lymph node revealed a thyroid transcription factor 1-positive (TTF-1) specimen concerning for lung adenocarcinoma in the absence of pulmonary nodules on imaging. Conclusions: Noteworthy to this case is the minimally invasive thoracoscopic approach preventing the need for median sternotomy and preventing any increased morbidity for the patient's incidentally found TxN3M0 lung adenocarcinoma. The patient could have been spared resection of the lymph node given its pulmonary origin as the standard of care for stage IIIB non-small cell lung cancer is definitive chemoradiation and adjuvant immunotherapy.

Ten-year Thyroid Cancer Incidence in an Integrated Healthcare Delivery System.

INTRODUCTION: The incidence of papillary thyroid cancer (PTC) has increased in recent decades, but data from community-based settings are limited. This study characterizes PTC trends in a large, integrated healthcare system over 10 years. METHODS: The annual incidence of PTC (2006-2015) was examined among Kaiser Permanente Northern California adults aged 21 to 84 years using Cancer Registry data, including tumor size and stage. Incidence estimates were age-adjusted using the 2010 US Census. RESULTS: Of 2990 individuals newly diagnosed with PTC (76.8% female, 52.7% non-Hispanic White), 38.5% and 61.5% were aged < 45 and < 55 years, respectively. At diagnosis, 60.9% had PTC tumors ≤ 2 cm, 9.2% had tumors > 4 cm, and 66.1% had Stage I disease. The annual age-adjusted incidence of PTC increased from 9.4 (95% confidence interval [CI] = 8.1-10.7) to 14.5 (95% CI = 13.1-16.0) per 100,000 person-years and was higher for female patients than for male patients. Incidence tended to be higher in Asian/Pacific Islanders and lower in Black individuals. Increasing incidence was notable for Stage I disease (especially 2006-2012) and evident across a range of tumor sizes (3.0-4.6 for ≤ 1 cm, 2.5-3.5 for 1-2 cm, and 2.4-4.7 for 2-4 cm) but was modest for large tumors (0.9-1.5 for > 4 cm) per 100,000 person-years. DISCUSSION: Increasing PTC incidence over 10 years was most evident for tumors ≤ 4 cm and Stage I disease. Although these findings may be attributable to greater PTC detection, the increase across a range of tumor sizes suggests that PTC burden might also have increased.

The Adult Neck Mass: Predictors of Malignancy.

OBJECTIVE: Distinguishing benign from malignant adult neck masses can be challenging because data to guide risk assessment are lacking. We examined patients with neck masses from an integrated health system to identify patient and mass factors associated with malignancy. STUDY DESIGN: Retrospective cohort. SETTING: Kaiser Permanente Northern California. METHODS: The medical records of adults referred to otolaryngology in 2017 for a neck mass were evaluated. Bivariate and multivariable logistic regression analyses were performed. RESULTS: Malignancy was found in 205 (5.0%) of the cohort's 4103 patients. Patient factors associated with malignancy included sex, age, and race/ethnicity. Males had more than twice the odds of malignancy compared with females (adjusted odds ratio [aOR] = 2.38). Malignancy rates increased with age, ranging from 2.1% for patients younger than 40 years to 8.4% for patients 70 years or older. White non-Hispanic patients had 1.75 times the risk of malignancy compared with patients of other race/ethnicities. The percentage of patients with malignancy increased with increasing minimum mass dimension, from 3.0% in patients with mass size <1 cm to over 31% in patients with mass sizes 2 cm or larger (P < .0001). Imaging-based mass factors most highly predictive of malignancy included larger minimum mass dimension (≥1.5 cm vs <1.5 cm: aOR = 3.87), multiple masses (2 or more vs 1: aOR = 5.07), and heterogeneous/ill-defined quality (aOR = 2.57). CONCLUSION: Most neck masses referred to otolaryngology were not malignant. Increasing age, male sex, white non-Hispanic ethnicity, increasing minimum mass dimension, multiple neck masses, or heterogeneous architecture/ill-defined borders were associated with malignancy.

Discovery of a Covalent Inhibitor of KRASG12C (AMG 510) for the Treatment of Solid Tumors.

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).

Risk of Mortality between Untreated and Treated Papillary Thyroid Cancer: A Matched Cohort Analysis.

OBJECTIVES: To examine the association between treatment status and mortality risk among patients with papillary thyroid cancer (PTC). METHODS: We identified 3,679 adults with PTC. Thirty-one untreated patients were matched to 155 treated patients. Hazards ratios (HR) and 95% confidence intervals (CIs) were calculated to estimate all-cause and disease-specific mortality. A low-risk subgroup was analyzed for differences in all-cause mortality. RESULTS: The adjusted HRs (95% CIs) for all-cause mortality at 5 and 10 years were 4.2 (1.7-10.3) and 4.1 (1.9-9.4) and for disease- specific mortality were 14.1 (3.4-59.3) and 10.2 (2.9-36.4), respectively, for untreated versus treated patients. The adjusted HRs (95% CIs) for all- cause mortality was 0.7 (0.1-6.4) for low-risk untreated versus matched treated patients. CONCLUSIONS: Compared to treated patients, untreated PTC patients were at higher risk of death while low-risk untreated PTC patients had comparable rate of metastasis and no increased risk of all-cause mortality. Level of evidence: 3.

Understanding the natural history of papillary thyroid cancer: Case series.

BACKGROUND: Papillary thyroid cancer (PTC) incidence continues to rise. We describe the natural history of untreated PTC patients. METHODS: Retrospective case series of 31 untreated PTC patients. RESULTS: We identified 31 untreated patients from the Kaiser Permanente Cancer Registry with PTC from 1973 to 2010. Patients were categorized as low risk (n = 16), high risk (n = 12), or low risk but medically contraindicated for surgery (n = 3). At diagnosis, 7 (58.3%) in the high-risk group had cervical lymph node metastases and 5 (41.7%) had distant metastases, compared to none in the low-risk group. Among the latter, three (18.8%) patients developed tumor growth >3 mm and one (6.3%) developed regional lymph node metastases without distant metastases. The 10-year overall survival was 71% and 35% for the low-risk and high-risk groups, respectively. CONCLUSIONS: Patients with low-risk untreated PTC were less likely to develop new regional or distant metastases and had better overall survival than patients with high-risk untreated PTC. LEVEL OF EVIDENCE: 4.

Association of NCCN-Recommended Posttreatment Surveillance With Outcomes in Patients With HPV-Associated Oropharyngeal Squamous Cell Carcinoma.

IMPORTANCE: National Comprehensive Cancer Network (NCCN) guidelines recommend routine clinical follow-up as posttreatment surveillance for patients with head and neck cancer (HNC). Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV-associated OPSCC) is a unique subset of HNC, associated with fewer recurrences and improved survival. The utility of this guideline in this patient population is unknown. OBJECTIVE: To determine adherence to the NCCN clinical follow-up guideline, frequency of recurrence detection method, classified as symptom-directed, physician-detected, or imaging-detected, and survival benefit associated with adherence to the NCCN guideline. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study of patients with HPV-associated OPSCC diagnosed between January 1, 2011, and April 30, 2014, at a large integrated health care system. Multivariable analyses were conducted using the Cox proportional hazards regression model, with patient adherence to NCCN visit guidelines constructed as a time-dependent variable. All data analyses were complete on September 1, 2018. EXPOSURES: Posttreatment clinical and imaging surveillance. MAIN OUTCOMES AND MEASURES: Recurrence and overall survival. Secondary outcome was salvage therapy. RESULTS: Of the 233 study patients with HPV-associated OPSCC, the mean (SD) age at diagnosis was 60.5 (8.7) years; 201 (86.3%) were male, 189 (81.1%) were white, and 109 (46.8%) had a positive smoking history. Median follow-up time through recurrence or all-cause mortality was 4.5 years (interquartile range, 3.8-5.6). Patients demonstrated 83.0% (180 of 217) adherence to NCCN surveillance guidelines in year 1, 52.7% (106 of 201) in year 2, 73.4% (141 of 192) in year 3, 62.3% (96 of 154) in year 4, and 52.9% (45 of 85) in year 5. A total of 3358 clinical surveillance examinations were performed with 22 patients having recurrences. There were 10 symptom-directed, 1 physician-detected, and 11 imaging-detected recurrences. Of the symptom-directed recurrences, salvage therapy was attempted in 5; at the study end date, 1 was alive. Salvage neck dissection was attempted in the physician-detected recurrence; this patient subsequently died. All locoregional recurrences occurred within the first 2 years, and all salvageable recurrences within the first year. Adherence to NCCN guidelines was not protective against all-cause mortality in the multivariable Cox proportional hazards regression model (hazard ratio, 0.76; 95% CI, 0.28-2.05). CONCLUSIONS AND RELEVANCE: Among patients with HPV-associated OPSCC, clinical surveillance is of limited utility. Nearly all clinically detected recurrences were elicited by patient symptoms that prompted earlier presentation to the clinician. Adherence to the current schedule does not appear to confer survival advantage, and locoregional recurrences are almost never detected beyond 2 years. These findings support reduction of posttreatment clinical surveillance in this population.

Posttreatment surveillance PET/CT for HPV-associated oropharyngeal cancer.

BACKGROUND: Surveillance positron emission tomography-computed tomography (PET/CT) is commonly used for treatment assessment of radiation therapy in head and neck cancer. However, human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC) patients represent a unique subpopulation, for which the utility of surveillance PET/CT has not been well studied. METHODS: In this retrospective chart review comprising 233 HPV+OPSCC patients, we evaluated surveillance PET/CT for diagnostic accuracy, downstream clinical impact, and survival. RESULTS: Surveillance PET/CT demonstrated 100% negative predictive value and sensitivity, 59.9% specificity, and 13.4% positive predictive value. Surveillance PET/CT led to 90 imaging studies and 31 biopsies; 91.1% and 77.4% were negative for recurrence, respectively. Surveillance PET/CT led to meaningful salvage therapy in 1.6% of cases. PET/CT-detected recurrences did not have improved survival compared to clinically detected recurrences. CONCLUSION: For HPV+OPSCC patients, surveillance PET/CTs frequently lead to unnecessary testing and rarely to meaningful disease salvage. They have no demonstrated survival benefit and should be interpreted cautiously to prevent patient harm.

Diagnostic Pathway of Oral Cavity Cancer in an Integrated Health Care System.

CONTEXT: Survival for patients with oral cavity squamous cell carcinoma (OCSCC) has remained relatively stagnant despite advances in treatment. Few studies have examined why advanced-stage disease is diagnosed in 40% of patients with OCSCC nationally. OBJECTIVE: To characterize the diagnostic pathway of OCSCC in an integrated health care system. DESIGN: Retrospective study of patients with OCSCC (2007-2010). MAIN OUTCOME MEASURES: Referral patterns and demographic, clinical, and tumor characteristics associated with time to diagnosis (diagnostic interval). RESULTS: Of 247 patients, 167 (68%) had early-stage (I/II) disease, 86 (35%) were referred by dentists, and 70 (28%) had a history of premalignancy. The median time (interquartile range) from symptom onset to care sought from a primary care physician (patient interval), from primary care physician to otolaryngologist, and from otolaryngologist to diagnosis was 8.6 (4.0-25.8), 1.0 (0.6-3.1), 0.0 (0.0-3.0) weeks, respectively. These intervals did not differ by demographic characteristics, clinical factors, or tumor stage. Prolonged diagnostic intervals were observed among patients with premalignant lesions. CONCLUSION: The patient interval was the largest component of the total diagnostic interval. The subsequent professional workup proceeded relatively efficiently. Prolonged diagnostic interval in patients with premalignant lesions may reflect the natural history of malignant transformation rather than a delay in diagnosis. However, nearly one-fourth of these cases were diagnosed at an advanced stage; closer surveillance may represent an opportunity for diagnosis at an earlier stage. Surveillance for premalignant lesions and facilitating referrals from dentists may expedite the diagnosis and treatment of OCSCC. Further investigation is warranted.

The diagnostic pathway of oropharyngeal squamous cell carcinoma in a large U.S. healthcare system.

OBJECTIVE: To examine the current diagnostic pathway of oropharyngeal squamous cell carcinoma (OPSCC) and identify factors associated with time to diagnosis. METHODS: Retrospective cohort study of patients with OPSCC in an integrated healthcare system from January 1, 2013, to December 31, 2013. Patient demographics, tobacco and alcohol use, chief complaint, tumor stage, human papilloma virus (HPV) status, physician factors (diagnosis, antibiotic prescription, performance of endoscopic exam, biopsy), and time intervals were examined. Time variations by patient characteristics and physician practice were assessed. RESULTS: We identified 152 patients with OPSCC. Of those, 90% had stage III to IV disease. The cohort was largely male (85%), white (79%), with HPV-positive tumors (84%). Most common chief complaints were neck mass (52%) and sore throat (20%). Among those with neck a mass, 94% had HPV-positive tumors. Prescription of antibiotics was associated with longer time to first otolaryngology evaluation. Median time from symptom onset to first primary care physician (PCP) contact was 3.0 weeks; from PCP to otolaryngologist was 1.1 weeks, and from otolaryngologist to tissue diagnosis was 0.4 weeks. At the first otolaryngology visit, 82% underwent in-office flexible endoscopy and 58% had same-day biopsy, resulting in rapid time to tissue diagnosis. Diagnostic time intervals did not differ by HPV status. CONCLUSION: The overall diagnostic process was efficient, although initial antibiotic treatment resulted in longer time to first otolaryngology visit. Tumor HPV status was associated with presenting findings but not time to diagnosis. The variation in diagnostic delay time and impact on survival outcomes is unknown and merits further investigation. LEVEL OF EVIDENCE: 4. Laryngoscope, 1867-1873, 2018.

Nasopharyngeal Carcinoma Diagnostic Challenge in a Nonendemic Setting: Our Experience with 101 Patients.

INTRODUCTION: We studied the presenting symptoms, time intervals, and workup involved in the diagnosis of nasopharyngeal carcinoma in an integrated health care system. METHODS: A retrospective chart review of all patients with a nasopharyngeal carcinoma diagnosis between 2007 and 2010 at Kaiser Permanente Northern California. Main outcome measures included diagnostic time intervals, presenting symptoms, diagnostic accuracy of nasal endoscopy, imaging, and diagnosis at first otolaryngologist (Oto-HNS) visit. RESULTS: This study included 101 patients: 70 (70%) were of Chinese or of Southeast Asian descent. The median time intervals along the diagnostic pathway were symptom onset to primary care physician visit, 6.0 weeks; primary care physician to Oto-HNS, 2.4 weeks; Oto-HNS to pathologic diagnosis, 1.1 weeks; and diagnosis to treatment onset, 5.5 weeks. The most common presenting symptoms were otologic issues (41, 41%), neck mass (39, 39%), nasal issues (32, 32%), and headache/cranial neuropathy (16, 16%). A nasopharyngeal lesion was detected in 54 (53%) patients after the first Oto-HNS visit. Among the initial nasal endoscopy reports, 32 (32%) did not reveal a nasopharyngeal lesion; 32 (32%) initial imaging studies also did not reveal a nasopharyngeal lesion. There was no correlation between diagnostic delay and disease stage. CONCLUSION: Nasopharyngeal carcinoma presenting symptoms are extremely variable, and initial misdiagnosis is common. Median time from symptom onset to treatment was almost six months among patients studied. Nearly one-third of nasopharyngeal cancers were missed with nasal endoscopy and imaging. An understanding of the risk factors, presenting symptoms, and limitations associated with these diagnostic tests is necessary to support earlier detection of this insidious cancer.

Economic and Demographic Characteristics of Cerumen Extraction Claims to Medicare.

IMPORTANCE: Cerumen extractions are performed in a large portion of otolaryngology and head and neck surgery practices. The burden on the health care system of cerumen extractions is unknown and demographics have not been characterized at a population level. OBJECTIVE: To quantify the cost burden and health care burden of cerumen extraction among Medicare recipients in the United States. DESIGN, SETTING, AND PARTICIPANTS: A retrospective cross-sectional study was conducted of all cerumen disimpactions documented in the Centers for Medicare & Medicaid Services Provider Utilization and Payment database from January 1 to December 31, 2012. Data analysis was conducted from August 4, 2014, to July 24, 2015. EXPOSURE: Cerumen disimpaction. MAIN OUTCOMES AND MEASURES: Cerumen extractions were analyzed by state, medical or surgical specialty, reimbursement, and type of health care professional performing the extraction. RESULTS: The Centers for Medicare & Medicaid Services reimbursed $46.8 million for 1.3 million cerumen disimpactions in 2012 (mean, $35.38 per procedure). The mean reimbursement rate per cerumen disimpaction varied by state from $25.41 in Puerto Rico to to $40.24 in New Jersey. The percentage of Medicare beneficiaries receiving cerumen extractions per state ranged from 0.55% in Puerto Rico to 4.92% in New Jersey. California had the overall highest total number of cerumen disimpactions (n = 132 823). The majority of cerumen extractions were performed by otolaryngology-head and neck practitioners (67.60%), although internal medicine (32.66%) and family practice (33.87%) had a higher amount of practitioners performing the procedure. The majority of cerumen extractions are performed by physicians (90.53%). CONCLUSIONS AND RELEVANCE: Cerumen extraction is one of the most common procedures performed by otolaryngology health care professionals. Practice patterns and reimbursement rates vary greatly across the country.

X-ray structures of Drosophila dopamine transporter in complex with nisoxetine and reboxetine.

Most antidepressants elicit their therapeutic benefits through selective blockade of Na(+)/Cl(-)-coupled neurotransmitter transporters. Here we report X-ray structures of the Drosophila melanogaster dopamine transporter in complexes with the polycyclic antidepressants nisoxetine or reboxetine. The inhibitors stabilize the transporter in an outward-open conformation by occupying the substrate-binding site. These structures explain how interactions between the binding pocket and substituents on the aromatic rings of antidepressants modulate drug-transporter selectivity.

Long Term Treatment Results for Deep Infections of Total Knee Arthroplasty.

This study aims to identify the long-term outcomes of total knee arthroplasty (TKA) treated for deep infection. 3270 consecutive primary and 175 revision TKAs were followed prospectively. There were 39 deep infections (1.16%): 29 primary (0.9%) and 10 revision (5.7%) cases. Two-stage resection and re-implantation procedure was performed in 13 primary cases with 10/13 (77%) successfully resolved. Early (<1 month) Irrigation and Debridement (I&D) was performed in 16 primary cases with 100% success. Late (>4 months) I&D was performed in 6 cases with 5/6 (83.3%) successful. Infection following revision TKA resulted in poor outcomes with both two-stage (2/4 successful) and I&D (2/6 successful). Deep infection after primary TKA can be successfully resolved with I&D and appropriate antibiotic treatment in the early postoperative course.

Neurotransmitter and psychostimulant recognition by the dopamine transporter.

Na(+)/Cl(-)-coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. Here we determine X-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine, a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants d-amphetamine and methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters.

Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.

Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His8-tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI(-) (N-acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4-6 weeks.

Structural basis for action by diverse antidepressants on biogenic amine transporters.

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

X-ray structure of dopamine transporter elucidates antidepressant mechanism.

Antidepressants targeting Na(+)/Cl(-)-coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses.