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The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. We have refined crystallization conditions for KRAS169 Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.

Background Naloxone, a life-saving medication that reverses opioid overdoses, was available in the United States only by prescription until March 2023, when the federal government approved nasal-spray formulations for over the counter sales to expand access. We assessed the availability of naloxone in a sample of pharmacies across the state of Connecticut. Methods Between September 15 and November 24, 2024, trained community-based volunteers surveyed a convenience sample of pharmacies throughout the state, focusing on naloxone signage, availability, cost, and in-store location. Pharmacies were categorized into three groups: chain pharmacies, pharmacies within grocery stores, and independent pharmacies. Summary statistics for the full sample and the three subgroups were tabulated, and differences between groups were analyzed using Fisher’s exact tests. Results A total of 162 pharmacies across all Connecticut counties were evaluated. While naloxone was available in most pharmacies, it was predominantly kept behind the pharmacy counter (n = 111, 73.5%) or the general checkout counter (n = 46, 30.5%). Fewer than 20% of pharmacies (n = 29) had naloxone easily accessible on an aisle shelf. Pricing was often high (≥ $60), particularly in independent pharmacies (n = 7, 22.6%; p < 0.001). Additionally, fewer than 20% of pharmacies (n = 31) displayed signage related to naloxone availability, and all signage was exclusively in English. Conclusions Despite widespread availability, naloxone access was restricted by its in-store location, high cost, and inadequate signage. This highlights a notable discrepancy between naloxone availability and accessibility, suggesting a lag in the effective implementation of policy in intended settings.

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

Werner syndrome helicase (WRN) is a RecQ-family DNA helicase essential for genome maintenance and is a synthetic lethal target in microsatellite instability-high (MSI-H) cancers. Despite its therapeutic promise, the conformational dynamics that enable WRN to unwind DNA, and how inhibitors disrupt this activity, remains poorly understood. Here, we present crystal structures of apo WRN and WRN bound to single-stranded DNA (ssDNA), capturing key conformations in the helicase catalytic cycle. These structures reveal how WRN engages DNA through conserved polar and aromatic interactions, and how domain rearrangements, including an ordering of the aromatic-rich loop (ARL), drive directional translocation. Biochemical and biophysical data demonstrate how nucleotide and inhibitor binding remodel these conformations and suggest that known clinical inhibitors (HRO761 and VVD-133214) function by locking WRN in inactive, ‘off-DNA’ states. Resistance emerged rapidly in vitro, through acquired point mutations as well as altered WRN expression. Together, our findings provide a structural framework for the WRN structural cycle and support the development of next-generation ‘on-DNA’ inhibitors to overcome resistance. In this study, Kennedy et al. combine crystallography, biophysical measurements and biochemical assays to define a structural cycle for Werner syndrome helicase (WRN) and reveal how nucleotide binding and ssDNA engagement lead to conformational transitions.

In 2019, Connecticut launched an opioid overdose–monitoring program to provide rapid intervention and limit opioid overdose–related harms. The Connecticut Statewide Opioid Response Directive (SWORD)—a collaboration among the Connecticut State Department of Public Health, Connecticut Poison Control Center (CPCC), emergency medical services (EMS), New England High Intensity Drug Trafficking Area (HIDTA), and local harm reduction groups—required EMS providers to call in all suspected opioid overdoses to the CPCC. A centralized data collection system and the HIDTA overdose mapping tool were used to identify outbreaks and direct interventions. We describe the successful identification of a cluster of fentanyl-contaminated crack cocaine overdoses leading to a rapid public health response. On June 1, 2019, paramedics called in to the CPCC 2 people with suspected opioid overdose who reported exclusive use of crack cocaine after being resuscitated with naloxone. When CPCC specialists in poison information followed up on the patients’ status with the emergency department, they learned of 2 similar cases, raising suspicion that a batch of crack cocaine was mixed with an opioid, possibly fentanyl. The overdose mapping tool pinpointed the overdose nexus to a neighborhood in Hartford, Connecticut; the CPCC supervisor alerted the Connecticut State Department of Public Health, which in turn notified local health departments, public safety officials, and harm reduction groups. Harm reduction groups distributed fentanyl test strips and naloxone to crack cocaine users and warned them of the dangers of using alone. The outbreak lasted 5 days and tallied at least 22 overdoses, including 6 deaths. SWORD’s near–real-time EMS reporting combined with the overdose mapping tool enabled rapid recognition of this overdose cluster, and the public health response likely prevented additional overdoses and loss of life.

Does it have an interior and exterior separated by a border between? \"to see in the human soul a place [lieu, locus]-no longer to regard the human person otherwise than cohabited by the powers that can take possession of it: exterior powers, autonomous, those evoked by the Gospel-the ornate house that awaits the visit of demons and which, visited, is the parable of the soul of each among us; this mental topology . . . the pathos itself [being] only a topos\" [Monnoyer 35].

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

The prolyl oligopeptidase family enzymes are a group of medically significant proteins distributed in all kingdoms of life that have several unifying structural and functional features. Members of the family hydrolyse short peptides, normally no more than 30 amino acids long. This strict substrate specificity is regulated by β-propeller domains that restrict access to the active site. Conformational changes allow access to the active site by small peptides but block access to larger, structured peptides. Mechanistic details such as these were revealed when the structure of prolyl oligopeptidase was solved by X-ray crystallography in 1998. Since then, numerous crystal structures of family members have been solved, increasing understanding of the structure-function relationships of these important enzymes. Many members of the family are either proven or potential drug targets. As such, structural characterization of these enzymes and their interactions facilitates the design of pharmaceutical compounds. With this in mind, this research was undertaken, using X-ray crystallography to further understand the interactions between particular members of the prolyl oligopeptidase family and inhibitor compounds. Four specific, potent inhibitors were visualized in complex with the active site of prolyl oligopeptidase. All four inhibitors follow a similar template which was shown to be successful when one of them advanced to phase 3 clinical trials as a treatment for Alzheimer‟s disease. The structure of oligopeptidase B from Trypanosoma brucei, which is involved in cell invasion leading to Trypanosomiasis, was solved to 2.5Å, revealing a number of interesting features and enabling various avenues of study. Finally, a plant derived glutamyl endopeptidase known to be regulated by endogenous inhibitors was overexpressed, purified to a high degree and shown to be correctly folded and active. Establishing this procedure allows for the commencement of crystal trials, as well as study by other biophysical methods.

Whitehead’s challenge to the age of science is, at its deepest stratum, where “it hath no bottom,” more clinical than critical. The claim to reduce the characteristic powers of life to an accidental result of the physical laws governing its elements is not simply an error; it is a symptom of thementalillness that affects human nature generally, but is florid in modernity with its “murder” of God. For God too is a symptom, but that symptom served to tie the knot whose unraveling exposes the brain to mental chaos and delirium when it tries to figure out what

\"Ein Landarzt\" presents the enigma of a nonlinear, seemingly illogical, dreamlike text-with no dreamer to supply us the associations and continuities it lacks. We can go a long way, however, on the evidence provided by: 1) the story's own context, placement; 2) the grammatical relations, first person, tense, activity, and passivity; 3) the extraordinary phonetic-semantic density of the first sentence, its resonance through the text; 4) the verbal and narrative displacements and condensations indicating complexes of identity and emotion; 5) \"Oedipal structure\" as vicious circle; 6) the exclusive disjunction: marriage or writing. Finally we begin to see emerging-as we have come to expect with Kafka-the complex logic of his desire and its laws. Kafka does not \"think through\" his oneiric creations: he elaborates verbal and pictorial figures in and against a multi-centered field of forces, powers, subjects of the law and transsubjective \"becomings\" of desire.