New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified > 10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECT's ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.

Social isolation has been associated with many adverse health outcomes in older adults. We describe a phone call outreach program in which health care professional student volunteers phoned older adults, living in long-term care facilities and the community, at risk of social isolation during the COVID-19 pandemic. Conversation topics were related to coping, including fears or insecurities, isolation, and sources of support; health; and personal topics such as family and friends, hobbies, and life experiences. Student volunteers felt the calls were impactful both for the students and for the seniors, and call recipients expressed appreciation for receiving the calls and for the physicians who referred them for a call. This phone outreach strategy is easily generalizable and can be adopted by medical schools to leverage students to connect to socially isolated seniors in numerous settings.

The efficacies of all antibiotics against tuberculosis are eventually eroded by resistance. New strategies to discover drugs or drug combinations with higher barriers to resistance are needed. Previously, we reported the application of a large-scale chemical-genetic interaction screening strategy called PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) for the discovery of new Mycobacterium tuberculosis inhibitors, which resulted in the identification of the small molecule BRD-8000, an inhibitor of a novel target, EfpA [Nature201951772]. Leveraging the chemical genetic interaction profile of BRD-8000, we identified BRD-9327, another structurally distinct small molecule EfpA inhibitor. We show that the two compounds are synergistic and display collateral sensitivity because of their distinct modes of action and resistance mechanisms. High-level resistance to one increases the sensitivity to and reduces the emergence of resistance to the other. Thus, the combination of BRD-9327 and BRD-8000 represents a proof-of-concept for the novel strategy of leveraging chemical genetics in the design of antimicrobial combination chemotherapy in which mutual collateral sensitivity is exploited.

During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9 mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.

The efficacies of all antibiotics against tuberculosis are eventually eroded by resistance. New strategies to discover drugs or drug combinations with higher barriers to resistance are needed. Previously, we reported the application of a large-scale chemicalgenetic interaction screening strategy called PROSPECT to the discovery of new Mycobacterium tuberculosis inhibitors, which resulted in identification of the small molecule BRD-8000, an inhibitor of a novel target, EfpA. Leveraging the chemical genetic interaction profile of BRD-8000, we identified BRD-9327, another, structurally distinct small molecule EfpA inhibitor. We show that the two compounds are synergistic and display collateral sensitivity because of their distinct modes of action and resistance mechanisms. Highlevel resistance to one increases the sensitivity to and reduces the emergence of resistance to the other. Thus, the combination of BRD-9327 and BRD-8000 represents a proof-of-concept for the novel strategy of leveraging chemical-genetics in the design of antimicrobial combination chemotherapy in which mutual collateral sensitivity is exploited.

New antibiotics are needed to combat rising resistance, with new Mycobacterium tuberculosis (Mtb) drugs of highest priority. Conventional whole-cell and biochemical antibiotic screens have failed. We developed a novel strategy termed PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) in which we screen compounds against pools of strains depleted for essential bacterial targets. We engineered strains targeting 474 Mtb essential genes and screened pools of 100-150 strains against activity-enriched and unbiased compounds libraries, measuring > 8.5-million chemical-genetic interactions. Primary screens identified >10-fold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insight. We identified > 40 novel compounds targeting DNA gyrase, cell wall, tryptophan, folate biosynthesis, and RNA polymerase, as well as inhibitors of a novel target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating PROSPECT's ability to yield inhibitors against novel targets which would have eluded conventional drug discovery.