Structure-activity relationships of middle-size cyclic peptides, KRAS inhibitors derived from an mRNA display.

Cyclic peptides are attracting attention as therapeutic agents due to their potential for oral absorption and easy access to tough intracellular targets. LUNA18, a clinical KRAS inhibitor, was transformed-without scaffold hopping-from the initial hit by using an mRNA display library that met our criteria for drug-likeness. In drug discovery using mRNA display libraries, hit compounds always possess a site linked to an mRNA tag. Here, we describe our examination of the Structure-Activity Relationship (SAR) using X-ray structures for chemical optimization near the site linked to the mRNA tag, equivalent to the C-terminus. Structural modifications near the C-terminus demonstrated a relatively wide range of tolerance for side chains. Furthermore, we show that a single atom modification is enough to change the pharmacokinetic (PK) profile. Since there are four positions where side chain modification is permissible in terms of activity, it is possible to flexibly adjust the pharmacokinetic profile by structurally optimizing the side chain. The side chain transformation findings demonstrated here may be generally applicable to hits obtained from mRNA display libraries.

Development of Orally Bioavailable Peptides Targeting an Intracellular Protein: From a Hit to a Clinical KRAS Inhibitor.

Cyclic peptides as a therapeutic modality are attracting a lot of attention due to their potential for oral absorption and accessibility to intracellular tough targets. Here, starting with a drug-like hit discovered using an mRNA display library, we describe a chemical optimization that led to the orally available clinical compound known as LUNA18, an 11-mer cyclic peptide inhibitor for the intracellular tough target RAS. The key findings are as follows: (i) two peptide side chains were identified that each increase RAS affinity over 10-fold; (ii) physico-chemical properties (PCP) including Clog P can be adjusted by side-chain modification to increase membrane permeability; (iii) restriction of cyclic peptide conformation works effectively to adjust PCP and improve bio-activity; (iv) cellular efficacy was observed in peptides with a permeability of around 0.4 × 10-6 cm/s or more in a Caco-2 permeability assay; and (v) while keeping the cyclic peptide's main-chain conformation, we found one example where the RAS protein structure was changed dramatically through induced-fit to our peptide side chain. This study demonstrates how the chemical optimization of bio-active peptides can be achieved without scaffold hopping, much like the processes for small molecule drug discovery that are guided by Lipinski's rule of five. Our approach provides a versatile new strategy for generating peptide drugs starting from drug-like hits.

Radioprotection of mice by lactoferrin against irradiation with sublethal X-rays.

The influence of a host defense protein, lactoferrin (LF), contained in exocrine secretions such as milk, on radiation disorder was investigated. A total of 25 C3H/He mice in each of two groups were maintained with 0.1% LF-added and LF-free diets, respectively, for one month. The mice were then treated with single whole-body X-ray irradiation at a sublethal dose (6.8 Gy), and the survival rate after irradiation was investigated. The survival rate at 30 d after irradiation was relatively higher in the LF group than in the control group (LF-free), (85 and 62%, respectively). The body weight 15 d after X-ray irradiation was also significantly greater in the LF group than in the control group. The hemoglobin level and hematocrit value were higher in the LF group at 5 d before X-ray irradiation. Another 52 mice underwent whole-body X-ray irradiation at the sublethal dose (6.8 Gy), and then LF was intraperitoneally injected once at 4 mg/animal to half of them. The survival rate in LF-treated mice 30 d after irradiation was 92%, significantly higher than in mice treated with saline (50%) (P = 0.0012). In addition, LF showed hydroxyl radical scavenger activity in vitro. These findings suggest that LF may inhibit radiation damage.

[Anesthetic case report of a patient with chronic inflammatory demyelinating polyneuropathy].

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune disease characterized by progressive neurological distress and motor weakness in the legs and arms. We report a patient with CIDP who underwent thoracoscopic surgery under general anesthesia. A 43-year-old man was hospitalized for examination of unidentified fever, and PET detected accumulations in the inguinal and mediastinal lymph nodes. The inguinal lymph node biopsy could not reveal the cause, and he was scheduled for thoracoscopic mediastinal lymph node biopsy. He had been diagnosed CIDP by lower motor weakness and sensory disorder for five months, and underwent peritoneal dialysis for chronic renal failure over the past nine months. Anesthesia was induced with propofol, remifentanil, and high-dose sevoflurane. He could be intubated easily with a left-sided Broncho-Cath double-lumen tube with no muscle relaxants. Anesthesia was maintained by sevoflurane (1.5-1.7%) and remifentanil (0.10-0.15 microg x kg(-1) x min(-1)). After the operation, spontaneous respiration appeared immediately after discontinuing anesthetics. Endotracheal tube was removed because of the following data; Sp(O2), 99%; tidal volume, about 600 ml; respiratory rate, 12-15 min(-1); level of consciousness was good. Arterial blood gas determination at this time revealed pH, 7.418: Pa(CO2), 36.0 mmHg : Pa(O2), 329.3 mmHg under 8 l x min(-1) oxygen. The patient showed an uncomplicated postoperative course.

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells.

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 microM, 2.4 microM, and 4.8 microM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 microM, 3.6 microM, and 5.4 microM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.

Potential of dark-striped field mice, Apodemus agrarius coreae, for use as a biological radiation dosimeter for human environments.

This study examined the possibility of using striped field mice as a biological dosimeter or indicator for surveillance of the ecological effects of boundary radiation emitted by nuclear power plants. For this study, the external morphological characteristics and isoenzymic electrophoretypes of Korean domestic dark-striped field mice were studied after they were captured, controlled for reproduction, and their exact species were identified. In terms of morphological external characteristics, the dark-brown coat, dark back stripe, head-to-tail length, tail length, and ear length matched the taxonomical characteristics of dark-striped field mice. In terms of isoenzymic electrophoretypes, the analyses on I-lactate dehydrogenase, aspartate aminotransferase, and malate dehydrogenase revealed that one species of dark-striped field mice, called Apodemus agrarius coreae, was scattered throughout a wide range of habitats. On the other hand, after irradiating the A. a. coreae (0, 0.5, 1, 3, 5, and 7 gray [Gy]) to analyze their survival rate and frequency of micronuclei in peripheral polychromatic erythrocytes, their LD50/30 was approximately 5 Gy. Also, the mice that contained 1 or 3 Gy gained weight compared with those that contained 0.5 Gy. Moreover, those with 0.5 Gy and higher showed an increase in white blood cells and platelets as well as in sodium and creatinine. However, decreased concentrations of alkaline phosphatase, alanine animotransferase, calcium, phosphorus, and globulin were observed in the A. a. coreae after irradiation. The results of the study reveal that wild A. a. coreae mice have high potential as a biological monitoring system to determine radiation effects in human environments such as those within the vicinity of nuclear power plants.