Identification, structural, and biophysical characterization of a positive modulator of human Kv3.1 channels.

Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.

Multiple models for outbreak decision support in the face of uncertainty.

Policymakers must make management decisions despite incomplete knowledge and conflicting model projections. Little guidance exists for the rapid, representative, and unbiased collection of policy-relevant scientific input from independent modeling teams. Integrating approaches from decision analysis, expert judgment, and model aggregation, we convened multiple modeling teams to evaluate COVID-19 reopening strategies for a mid-sized United States county early in the pandemic. Projections from seventeen distinct models were inconsistent in magnitude but highly consistent in ranking interventions. The 6-mo-ahead aggregate projections were well in line with observed outbreaks in mid-sized US counties. The aggregate results showed that up to half the population could be infected with full workplace reopening, while workplace restrictions reduced median cumulative infections by 82%. Rankings of interventions were consistent across public health objectives, but there was a strong trade-off between public health outcomes and duration of workplace closures, and no win-win intermediate reopening strategies were identified. Between-model variation was high; the aggregate results thus provide valuable risk quantification for decision making. This approach can be applied to the evaluation of management interventions in any setting where models are used to inform decision making. This case study demonstrated the utility of our approach and was one of several multimodel efforts that laid the groundwork for the COVID-19 Scenario Modeling Hub, which has provided multiple rounds of real-time scenario projections for situational awareness and decision making to the Centers for Disease Control and Prevention since December 2020.

HPVsim: An agent-based model of HPV transmission and cervical disease.

In 2020, the WHO launched its first global strategy to accelerate the elimination of cervical cancer, outlining an ambitious set of targets for countries to achieve over the next decade. At the same time, new tools, technologies, and strategies are in the pipeline that may improve screening performance, expand the reach of prophylactic vaccines, and prevent the acquisition, persistence and progression of oncogenic HPV. Detailed mechanistic modelling can help identify the combinations of current and future strategies to combat cervical cancer. Open-source modelling tools are needed to shift the capacity for such evaluations in-country. Here, we introduce the Human papillomavirus simulator (HPVsim), a new open-source software package for creating flexible agent-based models parameterised with country-specific vital dynamics, structured sexual networks, and co-transmitting HPV genotypes. HPVsim includes a novel methodology for modelling cervical disease progression, designed to be readily adaptable to new forms of screening. The software itself is implemented in Python, has built-in tools for simulating commonly-used interventions, includes a comprehensive set of tests and documentation, and runs quickly (seconds to minutes) on a laptop. Performance is greatly enhanced by HPVsim's multiscale modelling functionality. HPVsim is open source under the MIT License and available via both the Python Package Index (via pip install) and GitHub (hpvsim.org).

No antagonism or cross-resistance and a high barrier to the emergence of resistance in vitro for the combination of islatravir and lenacapavir.

Islatravir (ISL) is a deoxyadenosine analog that inhibits HIV-1 reverse transcription by multiple mechanisms. Lenacapavir (LEN) is a novel capsid inhibitor that inhibits HIV-1 at multiple stages throughout the viral life cycle. ISL and LEN are being investigated as once-weekly combination oral therapy for the treatment of HIV-1. Here, we characterized ISL and LEN in vitro to assess combinatorial antiviral activity, cytotoxicity, and the potential for interactions between the two compounds. Bliss analysis revealed ISL with LEN demonstrated additive inhibition of HIV-1 replication, with no evidence of antagonism across the range of concentrations tested. ISL exhibited potent antiviral activity against variants encoding known LEN resistance-associated mutations (RAMs) with or without the presence of M184V, an ISL RAM in reverse transcriptase (RT) . Static resistance selection experiments were conducted with ISL and LEN alone and in combination, initiating with either wild-type virus or virus containing the M184I RAM in RT to further assess their barrier to the emergence of resistance. The combination of ISL with LEN more effectively suppressed viral breakthrough at lower multiples of the compounds' IC50 (half-maximal inhibitory concentration) values and fewer mutations emerged with the combination compared to either compound on its own. The known pathways for development of resistance with ISL and LEN were not altered, and no novel single mutations emerged that substantially reduced susceptibility to either compound. The lack of antagonism and cross-resistance between ISL and LEN support the ongoing evaluation of the combination for treatment of HIV-1.

Covasim: An agent-based model of COVID-19 dynamics and interventions.

The COVID-19 pandemic has created an urgent need for models that can project epidemic trends, explore intervention scenarios, and estimate resource needs. Here we describe the methodology of Covasim (COVID-19 Agent-based Simulator), an open-source model developed to help address these questions. Covasim includes country-specific demographic information on age structure and population size; realistic transmission networks in different social layers, including households, schools, workplaces, long-term care facilities, and communities; age-specific disease outcomes; and intrahost viral dynamics, including viral-load-based transmissibility. Covasim also supports an extensive set of interventions, including non-pharmaceutical interventions, such as physical distancing and protective equipment; pharmaceutical interventions, including vaccination; and testing interventions, such as symptomatic and asymptomatic testing, isolation, contact tracing, and quarantine. These interventions can incorporate the effects of delays, loss-to-follow-up, micro-targeting, and other factors. Implemented in pure Python, Covasim has been designed with equal emphasis on performance, ease of use, and flexibility: realistic and highly customized scenarios can be run on a standard laptop in under a minute. In collaboration with local health agencies and policymakers, Covasim has already been applied to examine epidemic dynamics and inform policy decisions in more than a dozen countries in Africa, Asia-Pacific, Europe, and North America.

Preventing a cluster from becoming a new wave in settings with zero community COVID-19 cases.

BACKGROUND: In settings with zero community transmission, any new SARS-CoV-2 outbreaks are likely to be the result of random incursions. The level of restrictions in place at the time of the incursion is likely to considerably affect possible outbreak trajectories, but the probability that a large outbreak eventuates is not known. METHODS: We used an agent-based model to investigate the relationship between ongoing restrictions and behavioural factors, and the probability of an incursion causing an outbreak and the resulting growth rate. We applied our model to the state of Victoria, Australia, which has reached zero community transmission as of November 2020. RESULTS: We found that a future incursion has a 45% probability of causing an outbreak (defined as a 7-day average of > 5 new cases per day within 60 days) if no restrictions were in place, decreasing to 23% with a mandatory masks policy, density restrictions on venues such as restaurants, and if employees worked from home where possible. A drop in community symptomatic testing rates was associated with up to a 10-percentage point increase in outbreak probability, highlighting the importance of maintaining high testing rates as part of a suppression strategy. CONCLUSIONS: Because the chance of an incursion occurring is closely related to border controls, outbreak risk management strategies require an integrated approaching spanning border controls, ongoing restrictions, and plans for response. Each individual restriction or control strategy reduces the risk of an outbreak. They can be traded off against each other, but if too many are removed there is a danger of accumulating an unsafe level of risk. The outbreak probabilities estimated in this study are of particular relevance in assessing the downstream risks associated with increased international travel.

Discovery of macrocyclic HDACs 1, 2, and 3 selective inhibitors for HIV latency reactivation.

A series of unique macrocyclic HDACs 1, 2, and 3 selective inhibitors were identified with good enzymatic activity and high selectivity over HDACs 6 and 8. These macrocyclic HDAC inhibitors used an ethyl ketone as the zinc-binding group. Compounds 25 and 26 stood out as leads due to their low double-digit nM EC50s in the 2C4 cell-based HIV latency reactivation assay. The PK profiles of these macrocyclic HDAC inhibitors still needed improvement.

Modelling the impact of relaxing COVID-19 control measures during a period of low viral transmission.

OBJECTIVES: To assess the risks associated with relaxing coronavirus disease 2019 (COVID-19)-related physical distancing restrictions and lockdown policies during a period of low viral transmission. DESIGN: Network-based viral transmission risks in households, schools, workplaces, and a variety of community spaces and activities were simulated in an agent-based model, Covasim. SETTING: The model was calibrated for a baseline scenario reflecting the epidemiological and policy environment in Victoria during March-May 2020, a period of low community viral transmission. INTERVENTION: Policy changes for easing COVID-19-related restrictions from May 2020 were simulated in the context of interventions that included testing, contact tracing (including with a smartphone app), and quarantine. MAIN OUTCOME MEASURE: Increase in detected COVID-19 cases following relaxation of restrictions. RESULTS: Policy changes that facilitate contact of individuals with large numbers of unknown people (eg, opening bars, increased public transport use) were associated with the greatest risk of COVID-19 case numbers increasing; changes leading to smaller, structured gatherings with known contacts (eg, small social gatherings, opening schools) were associated with lower risks. In our model, the rise in case numbers following some policy changes was notable only two months after their implementation. CONCLUSIONS: Removing several COVID-19-related restrictions within a short period of time should be undertaken with care, as the consequences may not be apparent for more than two months. Our findings support continuation of work from home policies (to reduce public transport use) and strategies that mitigate the risk associated with re-opening of social venues.

Tenacious Endemic Typhoid Fever in Samoa.

BACKGROUND: Typhoid fever has been endemic on the island nation of Samoa (2016 population, 195 979) since the 1960s and has persisted through 2019, despite economic development and improvements in water supply and sanitation. METHODS: Salmonella enterica serovar Typhi isolates from the 2 hospitals with blood culture capability and matched patient demographic and clinical data from January 2008 through December 2019 were analyzed. Denominators to calculate incidence by island, region, and district came from 2011 and 2016 censuses and from 2017-2019 projections from Samoa's Bureau of Statistics. Data were analyzed to describe typhoid case burden and incidence from 2008 to 2019 by time, place, and person. RESULTS: In sum, 53-193 blood culture-confirmed typhoid cases occurred annually from 2008 to 2019, without apparent seasonality. Typhoid incidence was low among children age < 48 months (17.6-27.8/105), rose progressively in ages 5-9 years (54.0/105), 10-19 years (60.7-63.4/105), and 20-34 years (61.0-79.3/105), and then tapered off; 93.6% of cases occurred among Samoans < 50 years of age. Most typhoid cases and the highest incidence occurred in Northwest Upolu, but Apia Urban Area (served by treated water supplies) also exhibited moderate incidence. The proportion of cases from short-cycle versus long-cycle transmission is unknown. Samoan S. Typhi are pansusceptible to traditional first-line antibiotics. Nevertheless, enhanced surveillance in 2019 detected 4 (2.9%) deaths among 140 cases. CONCLUSIONS: Typhoid has been endemic in Samoa in the period 2008-2019. Interventions, including mass vaccination with a Vi-conjugate vaccine coadministered with measles vaccine are planned.

Development of a selective HDAC inhibitor aimed at reactivating the HIV latent reservoir.

The synthesis and SAR development of a trisubstituted imidazole HDAC inhibitor is described. The compounds were synthesized with high diastereocontrol by leveraging Ellman sulfinyl imine chemistry. Structural elucidation provided insight into binding mode and supported design rational. Pharmacokinetic properties of lead compounds were determined.

Discovery of ethyl ketone-based HDACs 1, 2, and 3 selective inhibitors for HIV latency reactivation.

A novel series of ethyl ketone based HDACs 1, 2, and 3 selective inhibitors have been identified with good enzymatic and cellular activity and high selectivity over HDACs 6 and 8. These inhibitors contain a spirobicyclic group in the amide region. Compound 13 stands out as a lead due to its good potency, high selectivity, and reasonable rat and dog PK. Compounds 33 and 34 show good potency and rat PK profiles as well.

Identification of potent inhibitors of the sortilin-progranulin interaction.

High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.

Augmenting Hit Identification by Virtual Screening Techniques in Small Molecule Drug Discovery.

Two orthogonal approaches for hit identification in drug discovery are large-scale in vitro and in silico screening. In recent years, due to the emergence of new targets and a rapid increase in the size of the readily synthesizable chemical space, there is a growing emphasis on the integration of the two techniques to improve the hit finding efficiency. Here, we highlight three examples of drug discovery projects at Merck & Co., Inc., Kenilworth, NJ, USA in which different virtual screening (VS) techniques, each specifically tailored to leverage knowledge available for the target, were utilized to augment the selection of high-quality chemical matter for in vitro assays and to enhance the diversity and tractability of hits. Central to success is a fully integrated workflow combining in silico and experimental expertise at every stage of the hit identification process. We advocate that workflows encompassing VS as part of an integrated hit finding plan should be widely adopted to accelerate hit identification and foster cross-functional collaborations in modern drug discovery.

Insights into activity and inhibition from the crystal structure of human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) catalyzes removal of ßα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.

Tuberculosis control strategies to reach the 2035 global targets in China: the role of changing demographics and reactivation disease.

BACKGROUND: In the last 20 years, China ramped up a DOTS (directly observed treatment, short-course)-based tuberculosis (TB) control program with 80% population coverage, achieving the 2015 Millennium Development Goal of a 50% reduction in TB prevalence and mortality. Recently, the World Health Organization developed the End TB Strategy, with an overall goal of a 90% reduction in TB incidence and a 95% reduction in TB deaths from 2015-2035. As the TB burden shifts to older individuals and China's overall population ages, it is unclear if maintaining the current DOTS strategy will be sufficient for China to reach the global targets. METHODS: We developed an individual-based computational model of TB transmission, implementing realistic age demographics and fitting to country-level data of age-dependent prevalence over time. We explored the trajectory of TB burden if the DOTS strategy is maintained or if new interventions are introduced using currently available and soon-to-be-available tools. These interventions include increasing population coverage of DOTS, reducing time to treatment, increasing treatment success, and active case finding among elders > 65 years old. We also considered preventative therapy in latently infected elders, a strategy limited by resource constraints and the risk of adverse events. RESULTS: Maintenance of the DOTS strategy reduces TB incidence and mortality by 42% (95% credible interval, 27-59%) and 41% (5-64%), respectively, between 2015 and 2035. A combination of all feasible interventions nears the 2035 mortality target, reducing TB incidence and mortality by 59% (50-76%) and 83% (73-94%). Addition of preventative therapy for elders would enable China to nearly reach both the incidence and mortality targets, reducing incidence and mortality by 84% (78-93%) and 92% (86-98%). CONCLUSIONS: The current decline in incidence is driven by two factors: maintaining a low level of new infections in young individuals and the aging out of older latently infected individuals who contribute incidence due to reactivation disease. While further reducing the level of new infections has a modest effect on burden, interventions that limit reactivation have a greater impact on TB burden. Tools that make preventative therapy more feasible on a large scale and in elders will help China achieve the global targets.

Structure of the bacterial deacetylase LpxC bound to the nucleotide reaction product reveals mechanisms of oxyanion stabilization and proton transfer.

The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.

Case Report: Pediatric Thyroid Storm Presenting to the Emergency Department with Afebrile Seizure.

Introduction: Seizures are a common presenting complaint and account for approximately 1% of total emergency department (ED) visits. Seizures are especially common in children less than five years old as they have a lower seizure threshold as compared to adults. One potentially dangerous etiology that is far less common, especially in children, is thyroid storm, the extreme manifestation of hyperthyroidism. Case Report: We describe the case of a 3-year-old girl who presented to the ED with an afebrile seizure but was found to be in thyroid storm. This case should serve as a reminder for emergency physicians to consider thyroid disease when evaluating patients presenting with seizures. Conclusion: Although most pediatric seizures are self-limited and frequently benign, it is imperative that the emergency physician evaluate for and rule out any potentially associated dangerous conditions such as thyroid storm.

Inferring the natural history of HPV from global cancer registries: insights from a multi-country calibration.

Human papillomavirus (HPV) is the cause of almost all cases of cervical cancer, a disease that kills some 340,000 women per year. The timeline from initial infection with HPV to the onset of invasive cervical cancer spans decades, and observational studies of this process are limited to settings in which treatment of precancerous lesions was withheld or inadequate. Such studies have been critical for understanding the natural history of HPV. Modeling can shed additional insight on the natural history of HPV, especially across geographical settings with varying prevalence of factors known to affect the host-side immune response to HPV, such as HIV and tobacco use. In this study, we create models for the 30 most populous countries in Sub-Saharan Africa, each with country-specific demographic, and behavioral inputs. We found that it was not possible to fit the data if we assumed that the natural history parameters were exactly identical for all countries, even after accounting for demographic and behavioral differences, but that we could achieve a good fit with the addition of a single immunocompetence parameter for each country. Our results indicate that variation in host immune responses may play a role in explaining the differences in the burden of cervical cancer between countries, which in turn implies a greater need for more geographically diverse data collection to understand the natural history of HPV.

Cerastecins inhibit membrane lipooligosaccharide transport in drug-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii infections have limited treatment options. Synthesis, transport and placement of lipopolysaccharide or lipooligosaccharide (LOS) in the outer membrane of Gram-negative bacteria are important for bacterial virulence and survival. Here we describe the cerastecins, inhibitors of the A. baumannii transporter MsbA, an LOS flippase. These molecules are potent and bactericidal against A. baumannii, including clinical carbapenem-resistant Acinetobacter baumannii isolates. Using cryo-electron microscopy and biochemical analysis, we show that the cerastecins adopt a serpentine configuration in the central vault of the MsbA dimer, stalling the enzyme and uncoupling ATP hydrolysis from substrate flipping. A derivative with optimized potency and pharmacokinetic properties showed efficacy in murine models of bloodstream or pulmonary A. baumannii infection. While resistance development is inevitable, targeting a clinically unexploited mechanism avoids existing antibiotic resistance mechanisms. Although clinical validation of LOS transport remains undetermined, the cerastecins may open a path to narrow-spectrum treatment modalities for important nosocomial infections.

Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.