Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain.

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

Cups for COVID: rapid implementation of a harm reduction initiative to support populations experiencing homelessness during the COVID-19 pandemic.

SETTING: As of June 10, 2020, 37 people experiencing homelessness or unstable housing in Calgary, Alberta, had developed lab-confirmed COVID-19. Spread occurred despite standard outbreak controls at affected shelter and supportive housing sites. Among these 37 cases, drink sharing was frequently identified as a modifiable mode of possible transmission. We collaborated with emergency shelters, a supportive housing site, and street and encampment outreach groups, using mixed service delivery by health staff, non-profits, and peers with lived experience with homelessness. INTERVENTION: To empower individuals to decrease COVID-19 transmission using a harm reduction approach, we provided disposable paper cups to service providers for distribution to clients. Service providers tracked the number of cups distributed. To assess effectiveness, we interviewed staff and peers who distributed the cups. OUTCOMES: Cup distribution was highest among populations with higher rates of alcohol use, and the intervention was well received by people who drink alcohol regularly, providing unique opportunities to promote COVID-19 awareness and safer drinking practices. Providers to these populations reported enthusiastic client engagement and repeat requests for cups for safer drinking. Intervention usefulness was limited in contexts with low alcohol consumption and in the absence of paired COVID-19 education. Provider reports suggest appropriate disposal of these cups after use. IMPLICATIONS: Disposable cups are a novel, rapidly implementable, low-cost harm reduction tool to empower people experiencing homelessness to reduce the risk of COVID-19 transmission due to drink sharing, ideally as part of a larger harm reduction and community education strategy.

RéSUMé: LIEU: Au 10 juin 2020, trente-sept (37) personnes sans abri ou vivant en logement instable à Calgary (Alberta) avaient contracté une infection par la COVID-19 confirmée en laboratoire. La maladie s'est propagée malgré les mesures types de contrôle des éclosions dans les refuges et les logements supervisés touchés. Parmi ces 37 cas, le partage de boissons a souvent été défini comme un mode de transmission modifiable possible. En collaboration avec des refuges d'urgence, un complexe de logements supervisés et des groupes menant des activités de proximité dans la rue et les campements, nous avons assuré une prestation de services mixte par des personnels de santé, des organisations sans but lucratif et des pairs ayant une expérience vécue de sans-abrisme. INTERVENTION: Pour donner à chaque personne les moyens de réduire la transmission de la COVID-19 selon une approche de réduction des méfaits, nous avons fourni aux dispensateurs de services des gobelets en papier jetables à distribuer à leurs usagers. Les dispensateurs ont fait un suivi du nombre de gobelets distribués. Pour évaluer l'efficacité de l'initiative, nous avons interviewé le personnel et les pairs ayant distribué les gobelets. RéSULTATS: Le nombre de gobelets distribués a été le plus élevé dans les populations ayant des taux élevés de consommation d'alcool, et l'intervention a été bien accueillie par les personnes qui consomment régulièrement de l'alcool; elle a offert des occasions uniques de faire de la sensibilisation à la COVID-19 et de promouvoir une pratique de consommation de boissons à moindre risque. Les intervenants auprès de ces populations ont fait état d'une participation enthousiaste des usagers et de demandes répétées de gobelets pour boire sans s'exposer au risque de contracter la maladie. L'utilité de l'intervention a été limitée dans les contextes de faible consommation d'alcool et en l'absence d'une sensibilisation conjointe à la COVID-19. Selon les rapports des dispensateurs de services, les gobelets ont été correctement éliminés après usage. CONSéQUENCES: Les gobelets jetables sont un nouvel outil de réduction des méfaits à prix abordable qui peut être mis en œuvre rapidement pour donner aux personnes aux prises avec le sans-abrisme les moyens de réduire le risque de transmission de la COVID-19 lorsqu'elles partagent des boissons, idéalement dans le cadre d'une stratégie de réduction des méfaits et de sensibilisation de proximité.

Small-molecule ligands of methyl-lysine binding proteins: optimization of selectivity for L3MBTL3.

Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small-molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug-discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small-molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure-activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells.

Histone recognition by human malignant brain tumor domains.

Histone methylation has emerged as an important covalent modification involved in a variety of biological processes, especially regulation of transcription and chromatin dynamics. Lysine methylation is found in three distinct states (monomethylation, dimethylation and trimethylation), which are recognized by specific protein domains. The malignant brain tumor (MBT) domain is one such module found in several chromatin regulatory complexes including Polycomb repressive complex 1. Here, we present a comprehensive characterization of the human MBT family with emphasis on histone binding specificity. SPOT-blot peptide arrays were used to screen for the methyllysine-containing histone peptides that bind to MBT domains found in nine human proteins. Selected interactions were quantified using fluorescence polarization assays. We show that all MBT proteins recognize only monomethyllysine and/or dimethyllysine marks and provide evidence that some MBT domains recognize a defined consensus sequence while others bind in a promiscuous, non-sequence-specific manner. Furthermore, using structure-based mutants, we identify a triad of residues in the methyllysine binding pocket that imparts discrimination between monomethyllysine and dimethyllysine. This study represents a comprehensive analysis of MBT substrate specificity, establishing a foundation for the rational design of selective MBT domain inhibitors that may enable elucidation of their role in human biology and disease.