Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Respiratory Auscultation Lab Using a Cardiopulmonary Auscultation Simulation Manikin.

Introduction: Mastery of respiratory auscultation skills is fundamental for clinicians to develop. We created a case-based educational session utilizing a high-fidelity simulator to teach lung sound auscultation to medical students at our institution. We employed a hypothesis-driven approach and deliberate practice to enhance students' learning experience and retention of acquired skills. Methods: We developed the session to teach second-year medical students how to discriminate between normal and pathological respiratory sounds within the context of clinical vignettes. Faculty facilitators, in conjunction with near-peer educators, made use of a high-fidelity auscultation manikin to guide students through case-based problem sets. Students were given the opportunity to auscultate the manikin while being observed and receiving feedback from the faculty. Results: We introduced the manikin in 2016, with a total of 759 second-year medical students from four class years having participated in the session since then. Students evaluated the session through an end-of-the-week and end-of-unit survey. The survey showed an overall improvement in learner satisfaction over previous years. Survey results and feedback were used to make adjustments to the session. Discussion: Our respiratory auscultation session was well received overall. Proper faculty development is crucial for implementing the session. Because of the focus on deliberate practice, adequate time must be allotted to hold the session. This program is reproducible with similar high-fidelity simulators.

Improving SCD compliance in trauma patients at Kings County Hospital Center: a quality improvement report.

Venous thromboembolism (VTE) is the fourth most commonly reported complication in trauma patients. For these patients, thromboprophylaxis is a standard of care. Patient compliance with sequential compression devices (SCDs), a form of mechanical VTE prophylaxis, has been a focus of efforts to improve patient safety. At our institution, a baseline audit in July 2020 revealed that patients admitted to the trauma floors have poor compliance with the use of SCDs. In this quality improvement project, we developed a patient education intervention to improve SCD compliance. We distributed an informational flyer to patients and led short educational sessions on VTE risk factors and proper SCD use. Our aim was to increase our SCD compliance rate by 30% in 4 weeks. We used three plan-do-study-act (PDSA) cycles to implement and refine our intervention. We measured SCD compliance during morning and afternoon patient observations and generated run charts to understand how our cycles were leading to change. After a 4-week period, we did not achieve our aim, but increased our overall compliance from 45% to 60% and sustained this improvement throughout our PDSA cycles. Morning compliance was lower than afternoon compliance both at baseline (45% vs 48.5%) and at the end the project (45% vs 53%). Our results suggest that patient education should be coupled with interventions that address other barriers to SCD compliance.

Discovery of Small Molecule Antagonists of the USP5 Zinc Finger Ubiquitin-Binding Domain.

USP5 disassembles unanchored polyubiquitin chains to recycle free monoubiquitin, and is one of the 12 ubiquitin specific proteases featuring a zinc finger ubiquitin-binding domain (ZnF-UBD). This distinct structural module has been associated with substrate positioning or allosteric modulation of catalytic activity, but its cellular function remains unclear. We screened a chemical library focused on the ZnF-UBD of USP5, crystallized hits in complex with the protein, and generated a preliminary structure-activity relationship, which enables the development of more potent and selective compounds. This work serves as a framework for the discovery of a chemical probe to delineate the function of USP5 ZnF-UBD in proteasomal degradation and other ubiquitin signaling pathways in health and disease.

Immunological memory to blood-stage malaria infection is controlled by the histamine releasing factor (HRF) of the parasite.

While most subunit malaria vaccines provide only limited efficacy, pre-erythrocytic and erythrocytic genetically attenuated parasites (GAP) have been shown to confer complete sterilizing immunity. We recently generated a Plasmodium berghei (PbNK65) parasite that lacks a secreted factor, the histamine releasing factor (HRF) (PbNK65 hrfΔ), and induces in infected mice a self-resolving blood stage infection accompanied by a long lasting immunity. Here, we explore the immunological mechanisms underlying the anti-parasite protective properties of the mutant PbNK65 hrfΔ and demonstrate that in addition to an up-regulation of IL-6 production, CD4+ but not CD8+ T effector lymphocytes are indispensable for the clearance of malaria infection. Maintenance of T cell-associated protection is associated with the reduction in CD4+PD-1+ and CD8+PD-1+ T cell numbers. A higher number of central and effector memory B cells in mutant-infected mice also plays a pivotal role in protection. Importantly, we also demonstrate that prior infection with WT parasites followed by a drug cure does not prevent the induction of PbNK65 hrfΔ-induced protection, suggesting that such protection in humans may be efficient even in individuals that have been infected and who repeatedly received antimalarial drugs.

Protection against malaria in mice is induced by blood stage-arresting histamine-releasing factor (HRF)-deficient parasites.

Although most vaccines against blood stage malaria in development today use subunit preparations, live attenuated parasites confer significantly broader and more lasting protection. In recent years, Plasmodium genetically attenuated parasites (GAPs) have been generated in rodent models that cause self-resolving blood stage infections and induce strong protection. All such GAPs generated so far bear mutations in housekeeping genes important for parasite development in red blood cells. In this study, using a Plasmodium berghei model compatible with tracking anti-blood stage immune responses over time, we report a novel blood stage GAP that lacks a secreted factor related to histamine-releasing factor (HRF). Lack of HRF causes an IL-6 increase, which boosts T and B cell responses to resolve infection and leave a cross-stage, cross-species, and lasting immunity. Mutant-induced protection involves a combination of antiparasite IgG2c antibodies and FcγR(+) CD11b(+) cell phagocytes, especially neutrophils, which are sufficient to confer protection. This immune-boosting GAP highlights an important role of opsonized parasite-mediated phagocytosis, which may be central to protection induced by all self-resolving blood stage GAP infections.

Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (Δhmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in Δhmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-γ(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria.

Plasmodium berghei histamine-releasing factor favours liver-stage development via inhibition of IL-6 production and associates with a severe outcome of disease.

Plasmodium spp., which causes malaria, produces a histamine-releasing factor (HRF), an orthologue of mammalian HRF. Histamine-releasing factor produced by erythrocytic stages of the parasite is thought to play a role in the pathogenesis of severe malaria. Here, we show in a rodent model that HRF is not important during the erythrocytic but pre-erythrocytic phase of infection, which mainly consists in the transformation in the liver of the mosquito-injected parasite form into the erythrocyte-infecting form. Development of P. berghei ANKA cl15cy1 liver stages lacking HRF is impaired and associated with an early rise in systemic IL-6, a cytokine that strongly suppresses development of Plasmodium liver stages. The defect is rescued by injection of anti-IL-6 antibodies or infection in IL-6-deficient mice and parasite HRF is sufficient to decrease IL-6 synthesis, indicating a direct role of parasite HRF in reducing host IL-6. The target cells modulated by HRF for IL-6 production at early time points during liver infection are neutrophils. Parasite HRF is thus used to down-regulate a cytokine with anti-parasite activity. Our data also highlight the link between a prolonged transition from liver to blood-stage infection and reduced incidence of experimental cerebral malaria.