Parsonage, D. | Search bioRxiv

bioRxiv2022

Kinetic and Redox Characterization of KRAS G12C Inhibition

The development of mutant-selective inhibitors for the KRASG12C allele has generated considerable excitement. These KRASG12C inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain, motivating the present studies. Measurement of the C12 thiol pKa by NMR and an independent biochemical assay found a depressed pKa (relative to free cysteine) of 7.6 consistent with its susceptibility to chemical ligation. Using a novel and validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that ARS-853 addition at 5{degrees}C elicited both a rapid first phase (attributed to binding, yielding a Kd of 36.0 {+/-} 0.7 M), and a second, slower pH-dependent phase taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C thiol to sulfinic acid alters RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.

biochemistry↗

bioRxiv2020

Acyl-lipid desaturases and Vipp1 cooperate in cyanobacteria to produce novel omega-3 PUFA-containing glycolipids

BackgroundDietary omega-3 (n-3), long chain (LC-, [≥] 20 carbons), polyunsaturated fatty acids (PUFAs) derived largely from marine animal sources protect against inflammatory processes and enhance brain development and function. With the depletion of natural stocks of marine animal sources and an increasing demand for n-3 LC-PUFAs, alternative, sustainable supplies are urgently needed. As a result, n-3 18 carbon and LC-PUFAs are being generated from plant or algal sources, either by engineering new biosynthetic pathways or by augmenting existing systems. ResultsWe utilized an engineered plasmid encoding two cyanobacterial acyl-lipid desaturases (DesB and DesD, encoding {Delta}15 and {Delta}6 desaturases, respectively) and "vesicle-inducing protein in plastids" (Vipp1) to induce production of stearidonic acid (SDA,18:4 n-3) at high levels in three strains of cyanobacteria (10, 17 and 27% of total lipids in Anabaena sp. PCC7120, Synechococcus sp. PCC7002, and Leptolyngbya sp. strain BL0902, respectively). Lipidomic analysis revealed that in addition to SDA, the rare anti-inflammatory n-3 LC-PUFA eicosatetraenoic acid (ETA, 20:4 n-3) was synthesized in these engineered strains, and [~]99% of SDA and ETA was complexed to bioavailable monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) species. Importantly, novel molecular species containing alpha-linolenic acid (ALA), SDA and/or ETA in both acyl positions of MGDG and DGDG were observed in the engineered Leptolyngbya and Synechococcus strains, suggesting that these could provide a rich source of anti-inflammatory molecules. ConclusionsOverall, this technology utilizes solar energy, consumes carbon dioxide, and produces large amounts of nutritionally-important n-3 PUFAs and LC-PUFAs. Importantly, it can generate previously-undescribed, highly bioavailable, anti-inflammatory galactosyl lipids. This technology could therefore be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products. Broader ContextDietary omega-3 (n-3), long chain polyunsaturated fatty acids (LC-PUFAs) typically found in marine products such as fish and krill oil are beneficial to human health. In addition to human consumption, most of the global supply of n-3 LC-PUFAs is used as dietary components for aquaculture. Marked increases in usage have created an intense demand for more sustainable, stable and bioavailable forms of n-3 PUFAs and LC-PUFAs. We utilized an engineered plasmid to dramatically enhance the production of 18-carbon and n-3 LC-PUFAs in three strains of autotrophic cyanobacteria. While the sustainable generation of highly valued and bioavailable nutritional lipid products is the primary goal, additional benefits include the generation of oxygen as a co-product with the consumption of only carbon dioxide as the carbon source and solar radiation as the energy source. This technology could be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products. Additionally, these engineered cyanobacteria can generate previously undescribed, highly bioavailable, anti-inflammatory galactosyl lipids.

molecular biology↗