Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo.

Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment. SIGNIFICANCE STATEMENT: We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings.

Death by a thousand cuts through kinase inhibitor combinations that maximize selectivity and enable rational multitargeting.

Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. However, the high sequence and structural conservation of the catalytic kinase domain complicate the development of selective kinase inhibitors. Inhibition of off-target kinases makes it difficult to study the mechanism of inhibitors in biological systems. Current efforts focus on the development of inhibitors with improved selectivity. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target effects. We develop a multicompound-multitarget scoring (MMS) method that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables optimization of inhibitor combinations for multiple on-targets. Using MMS with published kinase inhibitor datasets we determine potent inhibitor combinations for target kinases with better selectivity than the most selective single inhibitor and validate the predicted effect and selectivity of inhibitor combinations using in vitro and in cellulo techniques. MMS greatly enhances selectivity in rational multitargeting applications. The MMS framework is generalizable to other non-kinase biological targets where compound selectivity is a challenge and diverse compound libraries are available.

Probing conformational dynamics to understand kinase inhibition.

Why do some inhibitors select the on-state in ERK2, a kinase that is involved in many signaling pathways in cells, whereas others bind to more than one conformation?

Allosteric regulation and inhibition of protein kinases.

The human genome encodes more than 500 different protein kinases: signaling enzymes with tightly regulated activity. Enzymatic activity within the conserved kinase domain is influenced by numerous regulatory inputs including the binding of regulatory domains, substrates, and the effect of post-translational modifications such as autophosphorylation. Integration of these diverse inputs occurs via allosteric sites that relate signals via networks of amino acid residues to the active site and ensures controlled phosphorylation of kinase substrates. Here, we review mechanisms of allosteric regulation of protein kinases and recent advances in the field.

Death by a Thousand Cuts â€" Combining Kinase Inhibitors for Selective Target Inhibition and Rational Polypharmacology.

Kinase inhibitors are successful therapeutics in the treatment of cancers and autoimmune diseases and are useful tools in biomedical research. The high sequence and structural conservation of the catalytic kinase domain complicates the development of specific kinase inhibitors. As a consequence, most kinase inhibitors also inhibit off-target kinases which complicates the interpretation of phenotypic responses. Additionally, inhibition of off-targets may cause toxicity in patients. Therefore, highly selective kinase inhibition is a major goal in both biomedical research and clinical practice. Currently, efforts to improve selective kinase inhibition are dominated by the development of new kinase inhibitors. Here, we present an alternative solution to this problem by combining inhibitors with divergent off-target activities. We have developed a multicompound-multitarget scoring (MMS) method framework that combines inhibitors to maximize target inhibition and to minimize off-target inhibition. Additionally, this framework enables rational polypharmacology by allowing optimization of inhibitor combinations against multiple selected on-targets and off-targets. Using MMS with previously published chemogenomic kinase inhibitor datasets we determine inhibitor combinations that achieve potent activity against a target kinase and that are more selective than the most selective single inhibitor against that target. We validate the calculated effect and selectivity of a combination of inhibitors using the in cellulo NanoBRET assay. The MMS framework is generalizable to other pharmacological targets where compound specificity is a challenge and diverse compound libraries are available.

Motivation to Impact: Medical Student Volunteerism in the COVID 19 Pandemic.

Objective: Volunteerism represents an important mechanism to promote resilience, empathy, and general well-being in medical students, a group that stands to benefit. Medical students report feelings of fatigue, burnout, exhaustion, and stress that correlates with poor academic performance, and significant decline in empathy over the 3rd year of both MD and DO programs. Volunteer motivations have been shown to mediate participant well-being. The relationship between medical student volunteer motivations and specific outcomes during the COVID-19 pandemic has not been addressed. Methods: We characterized features of medical student volunteers during the COVID-19 pandemic in 2020, including volunteering motivation using the Volunteer Functions Inventory, the types of activities in which they participated, and the physical, psychosocial, and emotional outcomes they experienced following volunteering. Results: Altruistic and humanitarian values-centric motivation predicts positive volunteering outcomes including increased resilience, ability to deal with disappointment and loss, and ability to cope with the COVID-19 pandemic. Values-centric motivation also increases volunteer empathy independent of educational stage. Values-centric participants were more likely to select volunteering activities with patient contact, which promotes student empathy and resilience. Conversely, career-centric motivation does not predict positive outcomes. These students are more likely to engage in research-oriented activities. Conclusions: The efficacy of integrating volunteerism into medical school curricula may be limited by professional pressure that manifests as career-oriented motivation. We propose that practical integration should promote altruistic and humanitarian values-centric participant orientation to the volunteering process, which is associated with enhanced recruitment, preservation of empathy, and additional positive volunteering outcomes of interest.

Discovery and molecular basis of subtype-selective cyclophilin inhibitors.

Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC50) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation.

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.