Irinotecan Hydrochloride (CPT-11) in Dialysis Patients with Gastrointestinal Cancer

We investigated changes in drug disposition and toxicities with CPT-11 in 15 dialysis patients with gastrointestinal cancers to clarify whether CPT-11 could be administered safely in such patients. For comparison, the same parameters were also investigated in 10 cancer patients not undergoing dialysis. Items investigated included (1) plasma concentrations of SN-38, SN-38G and CPT-11 at 0, 1, 12, 24, 36, 48 and 72h after administration, together with a comparison of mean AUC values for 3 dose levels of CPT-11 (50, 60 and 70mg/m2) in dialysis patients and controls;and (2) occurrence of adverse events. Several findings emerged from this study:(1) No significant difference was observed in the AUC for SN-38 or CPT-11 between the dialysis and control groups;(2) The AUC for SN-38G at each dose was significantly higher in dialysis patients;and (3) Grade 1-4 leucopenia was observed in 11 of the dialysis patients. One patient developed grade 4 leucopenia and died due to sepsis. Anorexia, diarrhea, nausea, alopecia and interstitial pneumonia occurred in 6 dialysis patients. We found changes in drug dispositions of CPT-11, SN-38 and SN-38G in dialysis patients, suggesting that hepatic excretion, especially that of SN-38G, was increased. No significant difference in occurrence of adverse events was observed between the 2 groups. This indicates that CPT-11 can be administered safely in patients on dialysis.</p

Structural biology relating to electron transfer

Most electron transfer proteins have cofactor molecules, such as heme, flavin, and iron-sulfur clusters. Electron transfer reactions are regulated by the redox states of the cofactors, and hydrogen atoms are often involved in the conversion of redox states. Therefore, it is important to obtain precise structure information including hydrogen atoms for understanding the electron transfer reactions. We have determined structures of some electron transfer proteins, cytochrome b5 (b5; binding a heme cofactor) and NADH-cytochrome b5 reductase (b5R; binding a FAD cofactor) at the resolutions higher than 1.0 Å by X-ray crystallography. Structure information of some hydrogen atoms were clarified by these X-ray analyses. On the other hand, the rest of the hydrogen positions are still ambiguous.Neutron crystallography is a powerful technique to obtain accurate positions of hydrogen atoms in protein structures. Recently, we have performed high-resolution neutron and X-ray crystal structure analyses of b5R. b5R catalyzes the electron transfer from two electron carriers of NADH to one electron carrier of b5 and participates in fatty acid synthesis, cholesterol synthesis, and xenobiotic oxidation as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, b5R also participates in the reduction of methemoglobin. We succeeded in data collection of b5R (oxidized form) at high resolutions, 1.40 Å (at iBIX in J-PARC) and 1.45 Å (at BIODIFF in FRM-II), under cryogenic conditions. We have observed a hydrogen bonding network from FAD to His49, which is the only polar residue in a cluster of hydrophobic residues on the surface near FAD, so it seems to be responsible for electron transfer to b5. In addition, we have determined high-resolution X-ray crystal structures of the reduced form of b5R using wild-type and T66V mutant. The electron density map of the NAD cofactor clearly displays NAD+ and NADH states in wild-type and mutant, respectively. The neutron and X-ray structure analyses provide information about the hydrogen transfer pathway in b5R.3rd QST International Symposiu

Structural basis of tandemly connected engrailed homeodomains for designing an array of DNA-binding proteins

Widely used genome-editing enzymes such as TALEN and CRISPR have a high molecular weight, which makes them difficult to be delivered to cells. To develop a novel genome-editing enzyme with a smaller molecular weight, we have focused on engrailed homeodomain protein (EHD). In our previous work, we have successfully created a novel DNA-binding protein by connecting two EHD domains with a linker (EHD2). The protein specifically recognizes the tandemly connected target site of the individual EHD’s target site in E.coli cells. The specific recognition for the tandem target sequence was achieved only when the conserved arginine 53 of the EHD was mutated to alanine in the tandem proteins ((EHD[R53A])2). The tandem proteins without that mutation mostly bound to sequences containing the target of the monomeric EHD. To reveal the molecular mechanisms for recognition of the tandem target site, we determined a crystal structure of ((EHD[R53A])2-DNA complex. We found that base-specific interactions of I47, K50 and N51 with DNA in the major groove observed in the wt were completely preserved in ((EHD[R53A])2. Together with the biological functional analysis, we conclude that ((EHD[R53A])2 realizes the precise recognition of the tandem target sites in cell by concurrently two individual EHD domains’ binding to the target sites.3rd QST International Symposiu

Structural basis of tandemly connected engrailed homeodomains for designing a DNA binding protein array.

Genome editing has become important tools not only for basic biological research but also clinical appreciations. Currently used systems such as TALEN and CRISPR/Cas9 have a potential limitation for further applications, which is caused by their large molecular weight. Therefore, a novel molecular scaffold to overcome their limitation has been desired. For this purpose, we selected the engrailed homeodomain (EHD), because the domain composed of only ∼60 amino acids can recognize 6 base pairs. In our previous work1, we have evaluated whether two domains connected with a several length of linkers (EHD2) can recognize the tandem target sites in E.coli cells, in order to clarify that EHD2 can work as an array, as other genome editing enzymes. The key finding was that the tandem target site was recognized by EHD2 when the conserved arginine 53 of the EHD was mutated to alanine in the tandem proteins ((R53A)2), while the tandem proteins without the mutation mainly bound to the monomeric site. To reveal the molecular mechanisms for recognition of the tandem target site, we determined a crystal structure of (R53A)2-DNA complex. We obtained 1.6 Å crystal structure of (R53A)2 bound to a 12 mer DNA, after trying co-crystallization of (R53A)2 with 12 to 22 bps of DNA. The individual EHDs adopt the typical homeodomain fold. One domain has base specific interactions with a cognate DNA, however, the other domain has weak interactions with the phosphates of DNA to work for crystal packing. A close-up view of the mutation site in the domain bound to cognate DNA and that of previously solved monomeric engrailed homeodomain2 (wt) are shown in Figure 1. We created R53A mutant to reduce binding affinity by losing the hydrogen bonds with the phosphate which is observed in the wild type structure. In the wild type EHD, base-specific interactions of I47, K50 and N51 with DNA in the major groove are important for affinity and specificity to DNA. We found that these interactions were completely preserved in (R53A)2. Together with the biochemical data, we conclude that (R53A)2 realizes the precise recognition of the tandem target site in cell by concurrent binding of the two individual EHD domains because the binding of the two domains properly compensates the decrease in affinity of the individual R53A EHD to the monomeric target DNA, but does not too much.International Symposium on Diffraction Structure Biology 201

Structural basis for an array of engrailed homeodomains

Small DNA binding proteins to target desired sequences have the potential to become a scaffold of molecular tools such as genome-editing. In our study, engrailed homeodomain (EHD) has been chosen and evaluated whether it can work as a molecular module connected with each other to recognize the longer target sequence. It was previously shown that two EHDs connected with a linker (EHD2) recognizes a target sequence twice as long as a single EHD in cells only when arginine 53 in each EHD in the tandem protein is mutated to alanine ((EHD[R53A])2). To investigate the recognition mechanism of (EHD[R53A])2, the crystal structure of the (EHD[R53A])2-DNA complex was determined at 1.6 Å resolution. The individual EHDs were found to adopt the typical homeodomain fold. Most importantly, the base-specific interactions in the major groove necessary for the affinity/specificity of wild-type EHD were preserved in (EHD[R53A])2. Bacterial assays confirmed that the base-specific interactions are retained under cellular conditions. These observations indicate that R53A mutation causes only a loss of the arginine-phosphate interaction at the protein-DNA interface, which reduces the DNA binding affinity compared with the wild-type. We therefore conclude that (EHD[R53A])2 precisely recognizes tandem target sites within cells, enabling the individual EHDs to concurrently bind to the target sites with modest binding affinity. This suggests that modulation of the binding activity of each EHD is vital for constructing a protein array that can precisely recognize a sequence with multiple target sites