Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC), but the emergence of resistance mutations restricts their efficacy. We previously showed that RAS, BRAF and EGFR mutant alleles, which appear in circulating tumor DNA (ctDNA) during EGFR blockade, decline upon therapy withdrawal. We hypothesized that monitoring resistance mutations in blood could rationally guide subsequent therapy with anti-EGFR antibodies. We report here the results of CHRONOS, an open-label, single-arm phase 2 clinical trial exploiting blood-based identification of RAS/BRAF/EGFR mutations levels to tailor a chemotherapy-free anti-EGFR rechallenge with panitumumab (ClinicalTrials.gov: NCT03227926; EudraCT 2016-002597-12). The primary endpoint was objective response rate. Secondary endpoints were progression-free survival, overall survival, safety and tolerability of this strategy. In CHRONOS, patients with tissue-RAS WT tumors after a previous treatment with anti-EGFR-based regimens underwent an interventional ctDNA-based screening. Of 52 patients, 16 (31%) carried at least one mutation conferring resistance to anti-EGFR therapy and were excluded. The primary endpoint of the trial was met; and, of 27 enrolled patients, eight (30%) achieved partial response and 17 (63%) disease control, including two unconfirmed responses. These clinical results favorably compare with standard third-line treatments and show that interventional liquid biopsies can be effectively and safely exploited in a timely manner to guide anti-EGFR rechallenge therapy with panitumumab in patients with mCRC. Further larger and randomized trials are warranted to formally compare panitumumab rechallenge with standard-of-care therapies in this patient setting.

SummaryA gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N-and C-terminal domains suggests an evolutionary model involving a plasmid-mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer-Villiger monooxygenase, allowed the recombinant bacteria to grow on long-and medium-chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium-chain alkanes overlapping AlkB and in the first step of sub-terminal oxidation of long-chain alkanes. It was also demonstrated that CYP116B5 is a self-sufficient cytochrome P450 consisting of a heme domain (aa 1-392) involved in the oxidation step of n-alkanes degradation, and its reductase domain (aa 444-758) comprising the NADPH-, FMN-and [2Fe2S]-binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.

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This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

Macaca fascicularis P450 2C20 shares 92% identity with human cytochrome P450 2C8, which is involved in the metabolism of more than 8% of all prescribed drugs. To date, only paclitaxel and amodiaquine, two substrate markers of the human P450 2C8, have been experimentally confirmed as M. fascicularis P450 2C20 drugs. To bridge the lack of information on the ligands recognized by M. fascicularis P450 2C20, in this study, a three-dimensional homology model of this enzyme was generated on the basis of the available crystal structure of the human homologue P450 2C8 using YASARA. The results indicated that 90.0%, 9.0%, 0.5%, and 0.5% of the residues of the P450 2C20 model were located in the most favorable, allowed, generously allowed, and disallowed regions, respectively. The root-mean-square deviation of the C-alpha superposition of the M. fascicularis P450 2C20 model with the Homo sapiens P450 2C8 was 0.074 Å, indicating a very high similarity of the two structures. Subsequently, the 2C20 model was used for in silico screening of 58 known P450 2C8 substrates and 62 inhibitors. These were also docked in the active site of the crystal structure of the human P450 2C8. The affinity of each compound for the active site of both cytochromes proved to be very similar, meaning that the few key residues that are mutated in the active site of the M. fascicularis P450 do not prevent the P450 2C20 from recognizing the same substrates as the human P450 2C8.