Using a Function-First "Scout Fragment"-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Using a function-first 'scout fragment'-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic 'scout fragments', which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.

Playing in the clinical decision support sandbox: tools and training for all.

Objectives: Introduce the CDS-Sandbox, a cloud-based virtual machine created to facilitate Clinical Decision Support (CDS) developers and implementers in the use of FHIR- and CQL-based open-source tools and technologies for building and testing CDS artifacts. Materials and Methods: The CDS-Sandbox includes components that enable workflows for authoring and testing CDS artifacts. Two workshops at the 2020 and 2021 AMIA Annual Symposia were conducted to demonstrate the use of the open-source CDS tools. Results: The CDS-Sandbox successfully integrated the use of open-source CDS tools. Both workshops were well attended. Participants demonstrated use and understanding of the workshop materials and provided positive feedback after the workshops. Discussion: The CDS-Sandbox and publicly available tutorial materials facilitated an understanding of the leading-edge open-source CDS infrastructure components. Conclusion: The CDS-Sandbox supports integrated use of the key CDS open-source tools that may be used to introduce CDS concepts and practice to the clinical informatics community.

Destination-Specific and Home Environment Condom Norms Influence Sexual Behavior During Travel.

Travelers may adapt HIV risk-reduction practices based on perceived destination-specific norms. We examined the association between perceived condom norms and condomless anal sex (CAS) during international and domestic travel and in the home environment among men who have sex with men. Men who traveled internationally in the past 12 months were recruited by respondent-driven sampling (N = 501). Not knowing destination-specific condom norms was significantly associated with less CAS during international travel and in the home environment but not during domestic travel. Perceiving home environment condom norms to expect use of condoms was significantly associated with less CAS during domestic but not international travel. Men were less likely to engage in CAS during international travel when destination-specific condom norms were unknown. Unfamiliarity with the environment and culture may influence some men to refrain from higher-risk behaviors. During domestic travel, some men appeared to apply home environment condom norms, which may be erroneous in some situations and pose an HIV risk.

Coding Disparity and Specificity during Emergency Department Visits after Transitioning to the Tenth Version of the International Classification of Diseases.

The purpose of this study was to examine coding changes using the International Classification of Diseases (ICD) after the transition from ICD-9 to ICD-10. We studied a national cohort of emergency department visits from the Veterans Health Administration (VHA) before and after the transition, focusing on coding disparity and coding specificity. The cohort accounted for 2 million emergency department visits by 1.2 million patients. There were no statistical differences between the groups with respect to demographics, comorbidities, diagnoses, or use of medical services. While ICD-10 offered significantly more codes as well as more specific coding options, the ICD-10 encounters continued to use a small number of codes, were less likely to use multiple codes, and did not consistently exploit the more unique codes to create more specific diagnoses. These findings within the VHA system corresponded to similar challenges that have been documented with Medicare claims and in the private sector.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Unfolding Mechanisms and Conformational Stability of the Dimeric Endophilin N-BAR Domain.

Endophilin, which is a member of the Bin-amphiphysin-Rvs (BAR) domain protein superfamily, contains a homodimeric N-BAR domain of a characteristic crescent shape. The N-BAR domain comprises a six-helix bundle and is known to sense and generate membrane curvature. Here, we characterize aspects of the unfolding mechanism of the endophilin A1 N-BAR domain during thermal denaturation and examine factors that influence the thermal stability of this domain. Far-UV circular dichroism (CD) spectroscopy was applied to monitor changes in the secondary structure above room temperature. The protein's conformational changes were further characterized through Foerster resonance energy transfer and cross-linking experiments at varying temperatures. Our results indicate that thermal unfolding of the endophilin N-BAR is (minimally) a two-step process, with a dimeric intermediate that displays partial helicity loss. Furthermore, a thermal shift assay and temperature-dependent CD were applied to compare the unfolding processes of several truncated versions of endophilin. The melting temperature of the N-BAR domain decreased when we deleted either the N-terminal H0 helix or the unstructured linker of endophilin. This result suggests that these intrinsically disordered domains may play a role in structurally stabilizing the functional N-BAR domain in vivo. Finally, we show that single-site mutations can also compromise endophilin's thermal stability.

A chemical genetics approach to examine the functions of AAA proteins.

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.

Improving the benefits of HIV testing and referrals in large household surveys through active linkages to care: lessons and recommendations from the Namibia population-based HIV impact assessment (NAMPHIA), 2017.

In household-based surveys that include rapid HIV testing services (HTS), passive referral systems that give HIV-positive participants information about how and where to access ART but minimal follow-up support from survey staff may result in suboptimal linkage. In the 2017 Namibia Population-based HIV Impact Assessment (NAMPHIA), we piloted a system of active linkage to care and ART (ALCART) that utilized the infrastructure of existing community-based partner organizations (CBPOs). All HIV-positive participants age 15-64 years not on ART were given standard passive referrals to ART plus the option to participate in ALCART. Cases were assigned to CBPOs in participants' localities. Healthcare workers from the CBPO's contacted cases and facilitated their linkage to facility-based ART. A total of 510 participants were eligible and consented to ALCART. The majority were new diagnoses (80.8%), while the remainder were previously diagnosed but not on ART (19.2%). Of the 510, 473 (92.7%) were successfully linked into care. Of these, all but one initiated ART. Our ALCART system used existing CBPOs and contributed to >90% linkage-to-care and >99% ART-initiation among linked participants in a large, nationally-representative survey. This approach can be used to improve the potential benefits of HTS in other large population-based surveys.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling.

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

Sensitive and Label-Free SERS Detection of Single-Stranded DNA Assisted by Silver Nanoparticles and Gold-Coated Magnetic Nanoparticles.

An efficient surface-enhanced Raman scattering (SERS) method combined with DNA ligation is demonstrated in this proof-of-concept study for the detection of the single-strand DNA associated with BRAF V600E mutation. Gold-coated magnetic nanoparticles with 6-mercaptopyridine-3-carboxylic acid (MPCA) as internal reference attached on the surface (MNP@SiO2@Au-MPCA) and silver nanoparticles with Raman reporter 4-mercaptobenzonic acid (4-MBA) on the surface (Ag@4-MBA) are used as the SERS substrates. Rationally designed DNA probes are conjugated to these two types of nanoparticles, respectively. The single-stranded DNA containing the BRAF V600E mutation is the target analyte, which would act as the substrate for MNP@SiO2@Au-MPCA and Ag@4-MBA to be linked together through ligation. After multiple cycles of DNA ligation, more Ag@4-MBA are brought to the surface of MNP@SiO2@Au-MPCA. The resulting nanoparticles are easily isolated by a magnet rapidly from the mixture and redispersed in aqueous solution for homogeneous SERS measurement. The detection sensitivity is improved by the enhancement of the SERS peaks of 4-MBA between the plasmonic nanoparticles, and the detection quantification is improved by the use of internal reference, MPCA, for signal normalization. The intensity ratio of 4-MBA/MPCA increases linearly in the 1-100 fmol range of the matched DNA (BRAF mutation). Different ratios of matched DNA in the background of a large number of the single-base mismatched DNA (BRAF normal) are used to mimic real samples, and the intensity ratios of 4-MBA/MPCA are linear in the 0.02-1% range of matched DNA/mismatched DNA. The high sensitivity and specificity of the method demonstrate its potential for clinical use.

High Levels of Glutaminase II Pathway Enzymes in Normal and Cancerous Prostate Suggest a Role in 'Glutamine Addiction'.

Abstract: Many tumors readily convert l-glutamine to α-ketoglutarate. This conversion is almost invariably described as involving deamidation of l-glutamine to l-glutamate followed by a transaminase (or dehydrogenase) reaction. However, mammalian tissues possess another pathway for conversion of l-glutamine to α-ketoglutarate, namely the glutaminase II pathway: l-Glutamine is transaminated to α-ketoglutaramate, which is then deamidated to α-ketoglutarate by ω-amidase. Here we show that glutamine transaminase and ω-amidase specific activities are high in normal rat prostate. Immunohistochemical analyses revealed that glutamine transaminase K (GTK) and ω-amidase are present in normal and cancerous human prostate and that expression of these enzymes increases in parallel with aggressiveness of the cancer cells. Our findings suggest that the glutaminase II pathway is important in providing anaplerotic carbon to the tricarboxylic acid (TCA) cycle, closing the methionine salvage pathway, and in the provision of citrate carbon in normal and cancerous prostate. Finally, our data also suggest that selective inhibitors of GTK and/or ω-amidase may be clinically important for treatment of prostate cancer. In conclusion, the demonstration of a prominent glutaminase II pathway in prostate cancer cells and increased expression of the pathway with increasing aggressiveness of tumor cells provides a new perspective on 'glutamine addiction' in cancers.

Ratiometric SERS detection of polycyclic aromatic hydrocarbons assisted by ß-cyclodextrin-modified gold nanoparticles.

A surface-enhanced Raman scattering (SERS) method is described for the determination of trace polycyclic aromatic hydrocarbons (PAHs) in the environment efficiently and economically. Detection sensitivity is improved by modifying gold nanoparticles (AuNPs) with 4-mercaptophenylboronic acid (4-MPBA) conjugated to ß-cyclodextrin (ß-CD) as a new method for ratiometric determination of PAHs in solution. Pyrene (with a Raman band at 580 cm-1) and anthracene (750 cm-1) were used as the model analytes, while 4-MPBA (1570 cm-1) was used as the internal reference to normalize the SERS signals. The intensity ratios of pyrene/4-MPBA increase linearly in the 2 to 10 nM pyrene concentration range, and the intensity ratios of anthracene/4-MPBA increase linearly in the 10 to 100 nM anthracene concentration range. The detection limits are 0.4 nM for pyrene and 4.4 nM for anthracene. This method was applied to the determination of the two analytes in soil sample extracts and the recoveries of pyrene (at levels of 236 ng∙g-1 and 170 ng∙g-1) and anthracene (334 ng∙g-1 and 510 ng∙g-1) agreed well with the results from GC-MS analyses. The good recovery rates (101.8% and 102.5% for pyrene and 106.4% and 101.7% for anthracene) confirmed the reliability of the method. Graphical abstract Schematic illustration of SERS signal enhancement of pyrene, as an example of polycyclic aromatic hydrocarbons, by ß-cyclodextrin modified gold nanoparticles.

Estimating the Population Size of Female Sex Workers in Three South African Cities: Results and Recommendations From the 2013-2014 South Africa Health Monitoring Survey and Stakeholder Consensus.

BACKGROUND: Robust population size estimates of female sex workers and other key populations in South Africa face multiple methodological limitations, including inconsistencies in surveillance and programmatic indicators. This has, consequently, challenged the appropriate allocation of resources and benchmark-setting necessary to an effective HIV response. A 2013-2014 integrated biological and behavioral surveillance (IBBS) survey from South Africa showed alarmingly high HIV prevalence among female sex workers in South Africa's three largest cities of Johannesburg (71.8%), Cape Town (39.7%), and eThekwini (53.5%). The survey also included several multiplier-based population size estimation methods. OBJECTIVE: The objective of our study was to present the selected population size estimation methods used in an IBBS survey and the subsequent participatory process used to estimate the number of female sex workers in three South African cities. METHODS: In 2013-2014, we used respondent-driven sampling to recruit independent samples of female sex workers for IBBS surveys in Johannesburg, Cape Town, and eThekwini. We embedded multiple multiplier-based population size estimation methods into the survey, from which investigators calculated weighted estimates and ranges of population size estimates for each city's female sex worker population. Following data analysis, investigators consulted civil society stakeholders to present survey results and size estimates and facilitated stakeholder vetting of individual estimates to arrive at consensus point estimates with upper and lower plausibility bounds. RESULTS: In total, 764, 650, and 766 female sex workers participated in the survey in Johannesburg, Cape Town, and eThekwini, respectively. For size estimation, investigators calculated preliminary point estimates as the median of the multiple estimation methods embedded in the IBBS survey and presented these to a civil society-convened stakeholder group. Stakeholders vetted all estimates in light of other data points, including programmatic experience, ensuring inclusion only of plausible point estimates in median calculation. After vetting, stakeholders adopted three consensus point estimates with plausible ranges: Johannesburg 7697 (5000-10,895); Cape Town 6500 (4579-9000); eThekwini 9323 (4000-10,000). CONCLUSIONS: Using several population size estimates methods embedded in an IBBS survey and a participatory stakeholder consensus process, the South Africa Health Monitoring Survey produced female sex worker size estimates representing approximately 0.48%, 0.49%, and 0.77% of the adult female population in Johannesburg, Cape Town, and eThekwini, respectively. In data-sparse environments, stakeholder engagement and consensus is critical to vetting of multiple empirically based size estimates procedures to ensure adoption and utilization of data-informed size estimates for coordinated national and subnational benchmarking. It also has the potential to increase coherence in national and key population-specific HIV responses and to decrease the likelihood of duplicative and wasteful resource allocation. We recommend building cooperative and productive academic-civil society partnerships around estimates and other strategic information dissemination and sharing to facilitate the incorporation of additional data as it becomes available, as these additional data points may minimize the impact of the known and unknown biases inherent in any single, investigator-calculated method.