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.

Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Multiferroic nanocomposite fabrication via liquid phase using anodic alumina template.

We report a novel and inexpensive fabrication process of multiferroic nanocomposite via liquid phase using an anodic alumina template. The sol-gel spin-coating technique was used to coat the template with ferrimagnetic CoFe2O4. By dissolving the template with NaOH aqueous solution, a unique nanotube array structure of CoFe2O4 was obtained. The CoFe2O4 nanotube arrays were filled with, and sandwiched in, ferroelectric BaTiO3 layers by a sol-gel spin-coating method to obtain the composite. Its multiferroicity was confirmed by measuring the magnetic and dielectric hysteresis loops.

Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

Design and evaluation of azaindole-substituted N-hydroxypyridones as glyoxalase I inhibitors.

We conducted a high throughput screening for glyoxalase I (GLO1) inhibitors and identified 4,6-diphenyl-N-hydroxypyridone as a lead compound. Using a binding model of the lead and public X-ray coordinates of GLO1 enzymes complexed with glutathione analogues, we designed 4-(7-azaindole)-substituted 6-phenyl-N-hydroxypyridones. 7-Azaindole's 7-nitrogen was expected to interact with a water network, resulting in an interaction with the protein. We validated this inhibitor design by comparing its structure-activity relationship (SAR) with that of corresponding indole derivatives, by analyzing the binding mode with X-ray crystallography and by evaluating its thermodynamic binding parameters.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

A Novel Three-Step Approach for Secure Splenectomy During Laparoscopic Total Gastrectomy for Gastric Cancer.

BACKGROUND: Splenectomy during total gastrectomy increases operative morbidity (Nakata et al. in Surgical endoscopy 7:1817-1822, 2015). Establishing a safe approach to laparoscopic splenectomy is one of the most urgent issues in the treatment of proximal advanced gastric cancer, which invades to the greater curvature (Kawamura et al. in Gastric Cancer 3:662-668, 2015). We developed a novel three-step procedure for splenectomy during laparoscopic total gastrectomy (LTG). METHODS: Splenectomy consisted of three steps. Step 1 (dorsal approach): The pancreatic tail and spleen were mobilized. This step delineates the dissection plane and the anatomy around the pancreatic tail. Step 2 (suprapancreatic approach): The suprapancreatic peritoneum was incised to fenestrate to the mobilized space. The no. 11d station was dissected. The inferior branch of the splenic artery was exposed. Step 3 (splenic hilum approach): The spleen was lifted up to straighten the splenic hilum. The aim was to prolong the splenic vasculature and enable the surgeon to transect splenic vasculatures easily despite their anatomical diversity. Division of the splenic branches promotes mobility of the pancreatic tail, enabling precise dissection and preservation of its blood supply. RESULTS: Of 45 patients with gastric cancer who underwent LTG, seven underwent concurrent splenectomy. In all cases, splenectomy was successfully accomplished. The median operation time, duration of splenectomy, blood loss, number of total retrieved lymph nodes, lymph node counts from stations 10 and 11d, and drain amylase levels on the third postoperative day were 382 min, 94 min, 30 ml, 51, 5, 5, and 158 IU/L, respectively. Postoperative morbidity more severe than Clavien-Dindo grade 2 occurred in one case, with no pancreas-related morbidity. No mortality or conversion occurred. CONCLUSIONS: This laparoscopic procedure allows adequate nodal dissection and safe splenectomy.

Laparoscopic transhiatal lymphadenectomy in the lower mediastinum for adenocarcinoma of the esophagogastric junction.

Laparoscopic transhiatal esophagogastrectomy is difficult because the lower mediastinum is so deeply located that the operative field is narrow and restricted by surrounding organs. Therefore, we performed lymphadenectomy with opening of the bilateral mediastinal pleura to maintain safety and obtain better exposure of lymph nodes and important organs. We will present our technique for laparoscopic lower mediastinal lymphadenectomy and reconstruction for cancer of the esophagogastric junction. Five abdominal ports were used. Retraction of the left lobe of the liver exposed the esophageal hiatus. A long, narrow gastric tube (3 cm wide) was formed, and regional abdominal lymph nodes (No. 1, 2, 3a, 7, 8a, 9, 19, and 20) were resected. The diaphragmatic hiatus was widely split and the opened bilateral mediastinal pleura enabled better exposure for lymph node dissection and reconstruction. The level where the inferior vena cava passed through the diaphragm into the chest was used as a landmark to identify supradiaphragmatic (No. 111) and lower thoracic paraesophageal nodes (No. 110), which were completely retrieved with this procedure. The posterior mediastinal nodes (No. 112pulR, 112pulL, and 112aoA) were also retrieved with bilateral opening of the mediastinal pleura and dissection of the inferior pulmonary ligaments. An esophagogastric tube anastomosis with pseudo-fornix was made with a no-knife linear stapler to prevent postoperative reflux esophagitis. This approach enabled safe and accurate laparoscopic lower mediastinal nodal dissection. With the advantage of a narrow gastric tube, the good working space made tension-free anastomosis possible.

Disruption of CRAF-mediated MEK activation is required for effective MEK inhibition in KRAS mutant tumors.

MEK inhibitors are clinically active in BRAF(V600E) melanomas but only marginally so in KRAS mutant tumors. Here, we found that MEK inhibitors suppress ERK signaling more potently in BRAF(V600E), than in KRAS mutant tumors. To understand this, we performed an RNAi screen in a KRAS mutant model and found that CRAF knockdown enhanced MEK inhibition. MEK activated by CRAF was less susceptible to MEK inhibitors than when activated by BRAF(V600E). MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting such complexes enhanced inhibition of CRAF-dependent ERK signaling. Newer MEK inhibitors target MEK catalytic activity and also impair its reactivation by CRAF, either by disrupting RAF-MEK complexes or by interacting with Ser 222 to prevent MEK phosphorylation by RAF.

Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity.

Tumors with mutant RAS are often dependent on extracellular signal-regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS.

Esophageal metastasis of breast carcinoma.

Esophageal metastasis from primary breast cancer is an unusual manifestation. We recently treated a patient with dysphagia, whose breast cancer had been treated in the distant past. A 70-year-old woman had been followed regularly in our outpatient clinic for 14 years after her primary breast cancer treatment, with no apparent tumor recurrence. After 2 years absence, she consulted our clinic with progressive dysphagia. Contrast esophagography and endoscopic examination with ultrasonography revealed a protruding submucosal tumor that was histopathologically diagnosed as esophageal metastasis of breast cancer. Radiation therapy involving a total of 60 Gy in combination with aromatase inhibitor was given. The patient's dysphagia was greatly relieved, concomitant with marked improvement of the stenotic lesion on imaging. Since treatment for recurrent breast cancer is generally palliative, systemic (chemo- and/or endocrine-) therapy in combination with radiotherapy is the first-line option for esophageal metastasis of breast cancer.