Probing RAS Function Using Monobody and NanoBiT Technologies.

Missense mutations in the RAS family of oncogenes (HRAS, KRAS, and NRAS) are present in approximately 20% of human cancers, making RAS a valuable therapeutic target (Prior et al., Cancer Res 80:2969-2974, 2020). Although decades of research efforts to develop therapeutic inhibitors of RAS were unsuccessful, there has been success in recent years with the entrance of FDA-approved KRAS-specific inhibitors to the clinic (Skoulidis et al.

Exploring switch II pocket conformation of KRAS(G12D) with mutant-selective monobody inhibitors.

The G12D mutation is among the most common KRAS mutations associated with cancer, in particular, pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over the G12D mutation in HRAS and NRAS. Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and α3 helix, and captured this pocket in the most widely open form reported to date.

Mutations in the α4-α5 allosteric lobe of RAS do not significantly impair RAS signaling or self-association.

Mutations in one of the three RAS genes (HRAS, KRAS, and NRAS) are present in nearly 20% of all human cancers. These mutations shift RAS to the GTP-loaded active state due to impairment in the intrinsic GTPase activity and disruption of GAP-mediated GTP hydrolysis, resulting in constitutive activation of effectors such as RAF. Because activation of RAF involves dimerization, RAS dimerization has been proposed as an important step in RAS-mediated activation of effectors.

Targeting the "undruggable" RAS with biologics.

RAS proteins represent critical drivers of tumor development and thus are the focus of intense efforts to pharmacologically inhibit these proteins in human cancer. Although recent success has been attained in developing clinically efficacious inhibitors to KRAS, there remains a critical need for developing approaches to inhibit additional mutant RAS proteins. A number of anti-RAS biologics have been developed which reveal novel and potentially therapeutically targetable vulnerabilities in oncogenic RAS.

Role of and in Susceptibility to Resazurin.

The phenoxazine dye resazurin exhibits bactericidal activity against the Gram-negative pathogens and . One resazurin derivative, resorufin pentyl ether, significantly reduces vaginal colonization by in a mouse model of infection. The narrow spectrum of bacteria susceptible to resazurin and its derivatives suggests these compounds have a novel mode of action.