SignificanceMutants of RAS are major oncogenes and occur in many human cancers, but efforts to develop drugs that directly inhibit the corresponding constitutively active RAS proteins have failed so far. We therefore focused on SOS1, the guanine nucleotide exchange factor (GEF) and activator of RAS. A combination of high-throughput and fragment screening resulted in the identification of nanomolar SOS1 inhibitors, which effectively down-regulate active RAS in tumor cells. In cells with wild-type KRAS, we observed complete inhibition of the RAS-RAF-MEK-ERK pathway. In a mutant KRAS cell line, SOS1 inhibition resulted in a reduction of phospho-ERK activity by 50%. Together, the data indicate that inhibition of GEFs may represent a viable approach for targeting RAS-driven tumors.

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDEd solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDEd is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and Ga i1 interact with PDEd and that, at least for H-Ras, the intact C-terminus is required. We suggest PDEd to be a speci®c soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDEd-mediated transport.

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.X-ray structure | glucose facilitator | human MFS transporter | cytochalasin B | GLUT inhibitor

We recently discovered that inhibition of the lipid peroxidase GPX4 can selectively kill cancer cells in a therapy-resistant state through induction of ferroptosis. Although GPX4 lacks a conventional druggable pocket, covalent small-molecule inhibitors are able to overcome this challenge by reacting with the GPX4 catalytic selenocysteine residue to eliminate enzymatic activity. Unfortunately, all currently-reported GPX4 inhibitors achieve their activity through reactive chloroacetamide groups. We demonstrate that such chloroacetamide-containing compounds are poor starting points for further advancement given their promiscuity, instability, and low bioavailability. Development of improved GPX4 inhibitors, including those with therapeutic potential, requires the identification of new electrophilic chemotypes and mechanisms of action that do not suffer these shortcomings. Here, we report our discovery that nitrile oxide electrophiles, and a set of remarkable chemical transformations that generates them in cells from masked precursors, provide an effective strategy for selective targeting of GPX4. Our results, which include structural insights, target engagement assays, and diverse GPX4-inhibitor tool compounds, provide critical insights that may galvanize development of improved compounds that illuminate the basic biology of GPX4 and therapeutic potential of ferroptosis induction. In addition, our discovery that nitrile oxide electrophiles engage in highly selective cellular interactions and are bioavailable in their masked forms may be relevant for targeting other currently undruggable proteins, such as those revealed by recent proteome-wide ligandability studies.