Anthropogenic disturbance favours generalist over specialist parasites in bird communities: Implications for risk of disease emergence
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Environmental Sciences & Ecology↗
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A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus
Introduction African Swine Fever (ASF) is a highly infectious disease of pigs, caused by African swine fever virus (ASFV). The lack of vaccines and drugs makes strict disinfection practices to be one of the main measurements to curb the transmission of ASF. Therefore, it is important to assess if all viruses are inactivated after disinfection or after long time exposure in their natural conditions. Currently, the infectivity of ASFV is determined by virus isolation and culture in a biosafety level 3 (BSL-3) laboratory. However, BSL-3 laboratories are not readily available, need skilled expertise and may be time consuming. Methods In this study, a Triton X-100 assisted PMAxx-qPCR method was developed for rapid assessment of infectious ASFV in samples. PMAxx, an improved version of propidium monoazide (PMA), can covalently cross-link with naked ASFV-DNA or DNA inside inactivated ASFV virions under assistance of 0.1% (v/v) TritonX-100, but not with ASFV-DNA inside live virions. Formation of PMAxx-DNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, the limit of detection of the PMAxx-qPCR assay was 2.32log 10 HAD 50 /mL of infectious ASFV. Testing different samples showed that the PMAxx-qPCR assay was effective to evaluate intact ASFV virions after treatment by heat or chemical disinfectants and in simulated samples such as swine tissue homogenate, swine saliva swabs, and environmental swabs. However, whole-blood and saliva need to be diluted before testing because they may inhibit the PCR reaction or the cross-linking of PMAxx with DNA. Conclusion The Triton X-100 assisted PMAxx-qPCR assay took less than 3 h from sample to result, offering an easier and faster way for assessing infectious ASFV in samples from places like pig farms and pork markets.
Liu, Huan↗
Drug discovery efforts at George Mason University
With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.
59 BASIC BIOLOGICAL SCIENCES↗
Heterogeneous estimations of non-pharmaceutical mitigation behavior during the COVID-19 pandemic
The COVID-19 pandemic highlighted the importance of human behavior in mitigating the spread of disease. Nonetheless, human behavior is often overlooked in models of disease spread, particularly by underutilizing real-world data. We address this by estimating probabilities that individuals engage in behaviors that influence SARS-CoV-2 transmission risk during the COVID-19 pandemic, between September 2020 and June 2022. These behaviors include wearing a mask, using public transportation, spending time with others, avoiding contact with others, and going to work. Our estimates account for the age and sex of individuals and are generated for every county in the United States. We utilized multiple open-source datasets and United States Census data to produce these estimates. Multiple datasets were used for validation, showing our estimates demonstrated comparable accuracy and robustness. Our estimates aid in understanding human behavior dynamics during the COVID-19 pandemic and could be used to inform monthly or longer-term behavior in simulations of COVID-19. Moreover, the methods presented can be applied to other behaviors and features for future simulations of infectious disease.
97 MATHEMATICS AND COMPUTING↗
Urban forms, physical activity and body mass index: a cross-city examination using ISS Earth Observation photographs
Johnson Space Center has archived thousands of astronauts acquired Earth images. Some spectacular images have been widely used in news media and in k-12 class room, but their potential utilizations in health promotion and disease prevention have relatively untapped. The project uses daytime ISS photographs to define city forms and links them to city or metropolitan level health data in a multicity context. Road connectivity, landuse mix and Shannon's information indices were used in the classification of photographs. In contrast to previous remote-sensing studies, which tend to focus on a single city or a portion of a city, this project utilized photographs of 39 U.S. cities. And in contrast to previous health-promotion studies on the built environment, which tend to rely on survey respondents' responses to evaluate road connectivity or mixed land use for a single study site, the project examined the built environments of multiple cities based on ISS photos. It was found that road connectivity and landuse mix were not statistically significant by themselves, but the composite measure of the Shannon index was significantly associated with physical activity, but not BMI. Consequently, leisure-time physical activity seems to be positively associated with the urban complexity scale. It was also concluded that unless they are planned or designed in advance, photographs taken by astronauts generally are not appropriate for a study of a single-site built environment nor are they appropriate for a study of infectious diseases at a local scale. To link urban built environment with city-wide health indicators, both the traditional nadir view and oblique views should be emphasized in future astronauts' earth observation photographs.
Lin, Ge↗
Next Generation Microbiology Requirements
As humans continue to explore deep into space, microorganisms will travel with them. The primary means to mitigate the risk of infectious disease are a combination of prudent spacecraft design and rigorous operational controls. The effectiveness of these methods are evaluated by microbiological monitoring of spacecraft, food, water, and the crew that is performed preflight, in-flight, and post-flight. Current NASA requirements associated with microbiological monitoring are based on culture-based methodology where microorganisms are grown on a semi-solid growth medium and enumerated. Subsequent identification of the organisms requires specialized labor and large equipment, which historically has been performed on Earth. Requirements that rely strictly on culture-based units limit the use of non-culture based monitoring technology. Specifically, the culture-based "measurement criteria" are Colony Forming Units (CFU, representing the growth of one microorganism at a single location on the agar medium) per a given volume, area, or sample size. As the CFU unit by definition is culture-based, these requirements limit alternative technologies for spaceflight applications. As spaceflight missions such as those to Mars extend further into space, culture-based technology will become difficult to implement due to the (a) limited shelf life of the culture media, (b) mass/volume necessary to carry these consumables, and (c) problems associated with the production of biohazardous material in the habitable volume of the spacecraft. In addition, an extensive amount of new knowledge has been obtained during the Space Shuttle, NASA-Mir, and International Space Station Programs, which gave direction for new or modified microbial control requirements for vehicle design and mission operations. The goal of this task is to develop and recommend a new set of requirements for vehicle design and mission operations, including microbiological monitoring, based upon "lessons learned" and new technology. During 2011, this study focused on evaluating potable water requirements by assembling a forum of internal and external experts from NASA, other federal agencies, and academia. Key findings from this forum included: (1) Preventive design and operational strategies should be stringent and the primary focus of NASA's mitigation efforts, as they are cost effective and can be attained with conventional technology. (2) Microbial monitoring hardware should be simple and must be able to measure the viability of microorganisms in a sample. Multiple monitoring technologies can be utilized as long as at the microorganisms being identified can also be confirmed as viable. (3) Evidence showing alterations in the crew immune function and microbial virulence complicates risk assessments and creates the need for very conservative requirements. (4) One key source of infectious agents will always be the crew, and appropriate preventative measures should be taken preflight. (5) Water systems should be thoroughly disinfected (sterilized if possible) preflight and retain a residual biocide throughout the mission. Future forums will cover requirements for other types of samples, specifically spaceflight food and environmental samples, such as vehicle air and vehicle and cargo surfaces. An interim report on the potable water forum has been delivered to the Human Research Program with a final report on the recommendations for all sample types being delivered in September 2013.
Ott, C. M.↗
2015 Space Radiation Standing Review Panel
The 2015 Space Radiation Standing Review Panel (from here on referred to as the SRP) met for a site visit in Houston, TX on December 8 - 9, 2015. The SRP met with representatives from the Space Radiation Element and members of the Human Research Program (HRP) to review the updated research plan for the Risk of Radiation Carcinogenesis Cancer Risk. The SRP also reviewed the newly revised Evidence Reports for the Risk of Acute Radiation Syndromes Due to Solar Particle Events (SPEs) (Acute Risk), the Risk of Acute (In-flight) and Late Central Nervous System Effects from Radiation Exposure (CNS Risk), and the Risk of Cardiovascular Disease and Other Degenerative Tissue Effects from Radiation (Degen Risk), as well as a status update on these Risks. The SRP would like to commend Dr. Simonsen, Dr. Huff, Dr. Nelson, and Dr. Patel for their detailed presentations. The Space Radiation Element did a great job presenting a very large volume of material. The SRP considers it to be a strong program that is well-organized, well-coordinated and generates valuable data. The SRP commended the tissue sharing protocols, working groups, systems biology analysis, and standardization of models. In several of the discussed areas the SRP suggested improvements of the research plans in the future. These include the following: It is important that the team has expanded efforts examining immunology and inflammation as important components of the space radiation biological response. This is an overarching and important focus that is likely to apply to all aspects of the program including acute, CVD, CNS, cancer and others. Given that the area of immunology/inflammation is highly complex (and especially so as it relates to radiation), it warrants the expansion of investigators expertise in immunology and inflammation to work with the individual research projects and also the NASA Specialized Center of Research (NSCORs). Historical data on radiation injury to be entered into the Watson “big data” study must be used with caution. The general scientific issues of reproducibility, details of experimental methods and data analysis from preclinical and basic research laboratories have been raised broadly over the last few years (not specific to this work) and indicate that caution must be applied in the ways these data are used. This pertains to preclinical data and also to phase 3 clinical trials in radiation oncology and medical oncology. Of course, appropriate use and analysis of these “big-data” sets also offer the potential of pinpointing limitations and extracting remaining useful information. Emphasis should be placed on the latter possibility. A key target is risk reduction from radiation exposure. Progress of the entire space program, now moving towards the Mars mission, requires timely answers to key components of human risk, which are known to be complex. Periodic review of progress should be conducted with additional resources directed into achieving critical milestones. Turning the long red bars to yellow and green (or for some risks such as CNS possibly to grey) must be high priority. That such progress will require new science and not engineering means that it should be viewed in a knowledge-based light. The technology-based aspects of engineering issues are certainly as important, however, science and knowledge-based problems are solved in a different way than engineering. Timelines for engineering are more predictable, while for science, progress can be methodical with occasional major incremental findings that can rapidly change the rate of progress. As opportunities for rapid incremental changes arise, periodic enhancement of investment is strongly recommended to enable such new knowledge to be quickly and efficiently exploited. Collaborations and linkages with National Institute of Allergy and Infectious Diseases (NIAID), the Biomedical Advanced Research and Development Authority (BARDA) and the Department of Defense (DoD) are in place and more are encouraged, where possible, with the radiation injury and medical countermeasure studies. This could include utilizing some of their animal model testing contracts to facilitate obtaining results using common platforms. Such approach will facilitate the comparison of results among laboratories, and will facilitate and accelerate the development of medical countermeasures. It is particularly noteworthy that the NASA Space Radiation Element is reaching out to the Multidisciplinary European Low Dose Initiative (MELODI) platform coordinating low dose radiation risk research, and to other international agencies that are studying low dose radiation effects in an effort to fill the void generated by the cancelation of the Department of Energy (DOE) low dose radiation program. While NASA is working actively with NIAID and BARDA to integrate their relevant findings of radiation mitigator investigations to NASA programs, the committee notes its disappointment that the United States currently lacks a dedicated low dose radiation program with clear mechanistic orientation and aimed at the quantification and mitigation of human radiation risk on Earth. This void gives to the NASA Space Radiation Program Element special societal value, but also makes its overall design more challenging.
Steinberg, Susan↗
Insights from 20 years of the Molecule of the Month
For 20 years, Molecule of the Month articles have highlighted the functional stories of 3D structures found in the Protein Data Bank (PDB). The PDB is the primary archive of atomic structures of biological molecules, currently providing open access to more than 150,000 structures studied by researchers around the world. The wealth of knowledge embodied in this resource is remarkable, with structures that allow exploration of nearly any biomolecular topic, including the basic science of genetic mechanisms, mechanisms of photosynthesis and bioenergetics, and central biomedical topics like cancer therapy and the fight against infectious disease. The central motivation behind the Molecule of the Month is to provide a user-friendly introduction to this rich body of data, charting a path for users to get started with finding and exploring the many available structures. The Molecule of the Month and related materials are updated regularly at the education portal PDB-101 (http://pdb101.rcsb.org/), offering an ongoing resource for molecular biology educators and students around the world.
59 BASIC BIOLOGICAL SCIENCES↗
A Review of Coccidioides Research, Outstanding Questions in the Field, and Contributions by Women Scientists
Abstract Purpose of Review Coccidioidomycosis is an infectious disease that gained clinical significance in the early 20th century. Many of the foundational contributions to coccidioidomycosis research, including the discovery of the fungal disease agent, Coccidioides spp., were made by women. We review recent progress in Coccidioides research and big questions remaining in the field, while highlighting some of the contributions from women. Recent Findings New molecular-based techniques provide a promising method for detecting Coccidioides, which can help determine the dominate reservoir host and ideal environmental conditions for growth. Genetic and genomic analyses have allowed an understanding of population structure, species level diversity, and evolutionary histories. We present a current, comprehensive genome list, where women contributed many of these entries. Several efforts to develop a coccidioidomycosis vaccine are underway. Summary Women continue to pioneer research on Coccidioides, including the relationships between the fungi and the environment, genetics, and clinical observations. Significant questions remain in the field of Coccidioides, including the main host reservoir, the relationships between genotypic and phenotypic variation, and the underlying cause for chronic clinical coccidioidomycosis cases.
59 BASIC BIOLOGICAL SCIENCES↗
Dielectrophoresis assisted rapid, selective and single cell detection of antibiotic resistant bacteria with G-FETs
Time-consuming, expensive and low sensitivity diagnostic methods used for monitoring bacterial infections lead to unnecessary or delays in prescription of the right antibiotic treatment. Determining an optimal clinical treatment requires rapid detection and identification of pathogenic bacteria and their sensitivity to specific antimicrobials. However, diagnostic devices that meet all of these criteria have proven elusive thus far. Graphene field effect transistors (G-FET) are a promising solution, since they are highly sensitive to chemical/biological modification, can have fast detection times and can be placed on different substrates. Here, by integrating specific peptide probes over G-FETs, we present a proof-of-concept study for species and strain specific label-free detection of clinical strains of pathogenic bacteria with high specificity and sensitivity. We found that pyrene-conjugated peptides immobilized on G-FETs were capable of detecting pathogenic Staphylococcus aureus at the single-cell level and discriminate against other gram-positive and gram-negative bacterial pathogens. A similar device was able to discriminate between antibiotic resistant and sensitive strains of Acinetobacter baumannii , suggesting that these devices can also be used for detecting antibiotic resistive pathogens. Furthermore, a new means of enhancing attachment, electric-field assisted binding, reduced the detection limit to 104 cells/ml and the detection time to below 5 minutes. Here, the combination of single step attachment, inexpensive production, rapid, selective and sensitive detection suggest G-FETs plus pyrene-conjugated peptides are a new platform for solving major challenges faced in point of care diagnostics to fight infectious diseases and antimicrobial resistance.
77 NANOSCIENCE AND NANOTECHNOLOGY↗
Structure of the Inmazeb cocktail and resistance to Ebola virus escape
Monoclonal antibodies can provide important pre- or post-exposure protection against infectious disease for those not yet vaccinated or in individuals that fail to mount a protective immune response after vaccination. Inmazeb (REGN-EB3), a three-antibody cocktail against Ebola virus, lessened disease and improved survival in a controlled trial. Here, we present the cryo-EM structure at 3.1 Å of the Ebola virus glycoprotein, determined without symmetry averaging, in a simultaneous complex with the antibodies in the Inmazeb cocktail. This structure allows the modeling of previously disordered portions of the glycoprotein glycan cap, maps the non-overlapping epitopes of Inmazeb, and illuminates the basis for complementary activities and residues critical for resistance to escape by these and other clinically relevant antibodies. We further provide direct evidence that Inmazeb protects against the rapid emergence of escape mutants, whereas monotherapies even against conserved epitopes do not, supporting the benefit of a cocktail versus a monotherapy approach.
59 BASIC BIOLOGICAL SCIENCES↗
COVID-19 dynamics across the US: A deep learning study of human mobility and social behavior
This paper presents a deep learning framework for epidemiology system identification from noisy and sparse observations with quantified uncertainty. The proposed approach employs an ensemble of deep neural networks to infer the time-dependent reproduction number of an infectious disease by formulating a tensor-based multi-step loss function that allows us to efficiently calibrate the model on multiple observed trajectories. The method is applied to a mobility and social behavior-based SEIR model of COVID-19 spread. The model is trained on Google and Unacast mobility data spanning a period of 66 days, and is able to yield accurate future forecasts of COVID-19 spread in 203 US counties within a time-window of 15 days. Interestingly, a sensitivity analysis that assesses the importance of different mobility and social behavior parameters reveals that attendance of close places, including workplaces, residential, and retail and recreational locations, has the largest impact on the effective reproduction number. Furthermore, the model enables us to rapidly probe and quantify the effects of government interventions, such as lock-down and re-opening strategies. Taken together, the proposed framework provides a robust workflow for data-driven epidemiology model discovery under uncertainty and produces probabilistic forecasts for the evolution of a pandemic that can judiciously provide information for policy and decision making. All codes and data accompanying this manuscript are available at https://github.com/PredictiveIntelligenceLab/DeepCOVID19.
60 APPLIED LIFE SCIENCES↗
AI-based language models powering drug discovery and development
The discovery and development of new medicines is expensive, time-consuming, and often inefficient, with many failures along the way. Powered by artificial intelligence (AI), language models (LMs) have changed the landscape of natural language processing (NLP), offering possibilities to transform treatment development more effectively. Here, we summarize advances in AI-powered LMs and their potential to aid drug discovery and development. We highlight opportunities for AI-powered LMs in target identification, clinical design, regulatory decision-making, and pharmacovigilance. We specifically emphasize the potential role of AI-powered LMs for developing new treatments for Coronavirus 2019 (COVID-19) strategies, including drug repurposing, which can be extrapolated to other infectious diseases that have the potential to cause pandemics. Finally, we set out the remaining challenges and propose possible solutions for improvement.
60 APPLIED LIFE SCIENCES↗
Different chemical scaffolds bind to L-phe site in Mycobacterium tuberculosis Phe-tRNA synthetase
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mt), is one of the deadliest infectious diseases. The rise of multidrug-resistant strains represents a major public health threat, requiring new therapeutic options. Bacterial aminoacyl-tRNA synthetases (aaRS) have been shown to be highly promising drug targets, including for TB treatment. These enzymes play an essential role in translating the DNA gene code into protein sequence by attaching specific amino acid to their cognate tRNAs. They have multiple binding sites that can be targeted for inhibitor discovery: amino acid binding pocket, ATP binding pocket, tRNA binding site and an editing domain. Recently we reported several high-resolution structures of M. tuberculosis phenylalanyl-tRNA synthetase (MtPheRS) complexed with tRNA Phe and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. Here, in this study, using Nucleic Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) we identified fragments that bind to MtPheRS and we determined crystal structures of their complexes with MtPheRS/tRNA Phe . All the binders interact with the L-Phe amino acid binding site. The analysis of interactions of the new compounds combined with adenylate analog structure provides insights for the rational design of antituberculosis drugs. The 3 ' arm of the tRNA Phe in all the structures was disordered with exception of one complex with D-735 compound. In this structure the 3' CCA end of the acceptor stem is observed in the editing domain of MtPheRS providing insights regarding the post-transfer editing activity of class II aaRS.
Gade, Priyanka [Univ. of Chicago, IL (United State↗
Enabling machine learning-ready HPC ensembles with Merlin
With the growing complexity of computational and experimental facilities, many scientific researchers are turning to machine learning (ML) techniques to analyze large scale ensemble data. With complexities such as multi-component workflows, heterogeneous machine architectures, parallel file systems, and batch scheduling, care must be taken to facilitate this analysis in a high performance computing (HPC) environment. Here, we present Merlin, a workflow framework to enable large ML-friendly ensembles of scientific HPC simulations. By augmenting traditional HPC with distributed compute technologies, Merlin aims to lower the barrier for scientific subject matter experts to incorporate ML into their analysis. As a producer–consumer workflow model, Merlin enables multi-machine, cross-batch job, dynamically allocated yet persistent workflows capable of utilizing surge-compute resources. Key features of Merlin are a flexible HPC-centric interface, low per-task overhead, multi-tiered fault recovery, and a hierarchical sampling algorithm that allows for $\mathscr{O}$(N) task execution and $\mathscr{O}$(N ln N) task queuing to ensembles of millions of tasks. In addition to Merlin’s design, we test the algorithm’s performance in an HPC center and demonstrate the ability to enqueue 40 million simulations in 100 s, with a 30 millisecond per-task overhead that is independent of ensemble size. Finally, we describe some example applications that Merlin has enabled on leadership-class HPC resources, such as the ML-augmented optimization of nuclear fusion experiments and the calibration of infectious disease models to study the progression of and possible mitigation strategies for COVID-19.
97 MATHEMATICS AND COMPUTING↗
Lignin - A green material for antibacterial application — A review
Lignin's antibacterial properties have become increasingly relevant due to the rise of microbial infectious diseases and antibiotic resistance. Lignin is capable of interacting electrostatically with bacteria and contains polyphenols that cause damage to their cell walls. These features make lignin a desirable material to exhibit antibacterial behavior. Therefore, lignin in antibacterial applications offers a novel approach to address the growing need for sustainable and effective antibacterial materials. Recent research has explored the incorporation of lignin in various biomedical applications, such as wound dressings, implants, and drug delivery systems, highlighting their potential as a sustainable alternative to synthetic antibacterial agents. Furthermore, the development of lignin-based nanomaterials with enhanced antimicrobial activity is an active area of research that holds great promise for the future. Here, in this review, we have provided a summary of how lignin can be incorporated into different forms, such as composite and non-composite synthesis of antibacterial agents and their performances. The challenges and future considerations are also discussed in this review article.
59 BASIC BIOLOGICAL SCIENCES↗
Structural basis of heme scavenging by the ChtA and HtaA hemophores in Corynebacterium diphtheriae
Corynebacterium diphtheriae causes diphtheria, a potentially fatal infectious disease that damages tissues in the upper respiratory tract. In order to proliferate, this pathogen acquires the essential nutrient iron from heme (iron-protoporphyrin IX) primarily found in human hemoglobin (Hb). C. diphtheriae secretes ChtA and HtaA hemophore proteins that bind ferric heme (hemin) via conserved region (CR) domains. Here, we demonstrate that their CR domains scavenge hemin after it is spontaneously released from Hb, and define the structural basis of hemin binding to ChtA and the N-terminal CR domain from HtaA by determining X-ray crystal structures of their protein-hemin complexes. Resonance Raman and electron paramagnetic resonance experiments demonstrate that the CR domains from ChtA and HtaA engage in pentacoordinate hemin binding through a conserved iron-tyrosyl linkage, though variations in their hemin pockets alter the way they stabilize the axial tyrosine and mask hemin’s metal. The importance of these interactions is probed using isothermal titration calorimetry experiments, which represent the first quantitative assessment of CR-hemin affinity and reveal that ChtA binds hemin via an enthalpically driven process. Hemin partitioning experiments using native mass spectrometry demonstrate that the cohort of CR domains within C. diphtheriae ’s hemin-uptake system have dissociation constants for hemin between 0.8 and 22 nM, raising the possibility that affinity differences contribute to the directional flow of hemin into the cell. Collectively, the results of this work provide insight into how C. diphtheriae and other pathogenic and commensal corynebacterium species utilize CR domains to scavenge iron rich hemin from their environment.
Corynebacterium diphtheriae↗
Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2)
Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here we use structure-based drug prototyping to identify two classes of promising leads that non-competitively inhibit UCK2 activity. In the process we have identified a hitherto unknown allosteric site at the inter-subunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.
59 BASIC BIOLOGICAL SCIENCES↗