Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.

Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

A new opecoelid species (Trematoda: Opecoelidae) from the threespine stickleback Gasterosteus aculeatus L. in California.

Plagioporus kolipinskii n. sp. (Trematoda: Opecoelidae) is described from the intestine of the threespine stickleback, Gasterosteus aculeatus L., from Lobos Creek, a freshwater stream in Presidio, San Francisco County, California. Plagioporus kolipinskii is morphologically somewhat similar to 4 (P. serotinus , P. angusticollis , P. macrouterinus , and P. shawi []) of the 12 currently recognized North American species of the genus, but can be readily distinguished from all 4 in possessing a much larger acetabulum and a larger ovary relative to the testes. In addition, the new species can be distinguished from P. angusticollis by a smaller cirrus sac; from P. macrouterinus by the elongated shape of its body and reduced extent of its uterus (the uterus of P. macrouterinus extends posteriorly to the intersection of the testes); from P. shawi by a much-shorter cirrus sac (which reaches the ovary in P. shawi), an unlobed ovary (as opposed to a quadrilobed ovary in P. shawi), and fewer eggs that are also larger relative to body size.