Peptide-to-Small Molecule: A Pharmacophore-Guided Small Molecule Lead Generation Strategy from High-Affinity Macrocyclic Peptides.

Recent technological innovations have led to the development of methods for the rapid identification of high-affinity macrocyclic peptides for a wide range of targets; however, it is still challenging to achieve the desired activity and membrane permeability at the same time. Here, we propose a novel small molecule lead discovery strategy, ″Peptide-to-Small Molecule″, which is a combination of rapid identification of high-affinity macrocyclic peptides via peptide display screening followed by pharmacophore-guided de novo design of small molecules, and demonstrate the applicability using nicotinamide N-methyltransferase (NNMT) as a target. Affinity selection by peptide display technology identified macrocyclic peptide 1 that exhibited good enzymatic inhibitory activity but no cell-based activity. Thereafter, a peptide pharmacophore-guided de novo design and further structure-based optimization resulted in highly potent and cell-active small molecule 14 (cell-free IC50 = 0.0011 µM, cell-based IC50 = 0.40 µM), indicating that this strategy could be a new option for drug discovery.

Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.

Discovery of Extremely Selective Fused Pyridine-Derived ß-Site Amyloid Precursor Protein-Cleaving Enzyme (BACE1) Inhibitors with High In Vivo Efficacy through 10s Loop Interactions.

ß-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is considered to be a promising target for treating Alzheimer's disease. However, all clinical BACE1 inhibitors have failed due to lack of efficacy, and some have even led to cognitive worsening. Recent evidence points to the importance of avoiding BACE2 inhibition along with careful dose titration. In this study, we focused on the fact that the 10s loop lining the S3 pocket in BACE1 can form both "open (up)" and "closed (down)" conformations, whereas in BACE2, it prefers to adopt a "closed" form; thus, more space is available in BACE1. By leveraging the difference, we designed fused pyridine analogues that could reach the 10s loop, leading to 6 with high selectivity and significant Aß reduction. The cocrystal structures confirmed that 6 significantly increased B-factors of the 10s loop in BACE2 relative to those in BACE1. Thus, the destabilization of BACE2 seems to offer structural insights into the reduced BACE2 potency of 6, explaining the significant improvement in BACE1 selectivity.

Mepolizumab decreased the levels of serum galectin-10 and eosinophil cationic protein in asthma.

BACKGROUND: Mepolizumab, a humanized antibody targeting interleukin-5, decreases the number of blood eosinophils and the frequency of exacerbation of severe asthma. Galectin-10 is a protein within the cytoplasm of eosinophils and constitutes Charcot-Leyden crystals, which promotes key features of asthma. However, the relationship between time kinetics and clinical response of eosinophil-derived molecules such as galectin-10 or eosinophil cationic protein (ECP) has not been precisely investigated. OBJECTIVE: This study aimed to clarify the precise time course of the levels of serum galectin-10 and ECP after mepolizumab treatment and to analyze the relationship between the levels of eosinophil-derived molecules and the clinical background or response to mepolizumab treatment. METHODS: This multicenter, prospective open-label study recruited 20 patients with severe eosinophilic asthma. Mepolizumab was administered every 4 weeks for 32 weeks and the levels of various biomarkers were serially analyzed. RESULTS: The serum galectin-10 and ECP significantly and rapidly decreased 4 weeks after initial administration of mepolizumab. In contrast, basophil count, fractional exhaled nitric oxide, and the serum total IgE level were unchanged during treatment. Asthma Control Questionnaire-5, Asthma Health Questionnaire-33, and Lund-Mackay scores significantly improved after mepolizumab treatment. Both high ECP and eosinophil count related to better response in forced expiratory volume in 1 second (FEV1) and measurable ECP level at 4 weeks after administration of mepolizumab related to the further improvement in FEV1 toward week 32. No significant difference in improvement in FEV1 was observed in galectin-10 high group. The level of ECP at baseline was significantly related to the higher prevalence of nasal polyp and Lund-Mackay score. CONCLUSION: This study was the first to show that the levels of serum galectin-10 decreases after initial administration of mepolizumab. The significant relationship between serum ECP and better response in FEV1 suggested the potential role of serum ECP as a predictive biomarker for the efficacy of mepolizumab (UMIN000030466).

Macrocyclic Peptides as a Novel Class of NNMT Inhibitors: A SAR Study Aimed at Inhibitory Activity in the Cell.

Nicotinamide N-methyltransferase (NNMT), which catalyzes the methylation of nicotinamide, is a cytosolic enzyme that has attracted much attention as a therapeutic target for a variety of diseases. However, despite the considerable interest in this target, reports of NNMT inhibitors have still been limited to date. In this work, utilizing in vitro translated macrocyclic peptide libraries, we identified peptide 1 as a novel class of NNMT inhibitors. Further exploration based on the X-ray cocrystal structures of the peptides with NNMT provided a dramatic improvement in inhibitory activity (peptide 23: IC50 = 0.15 nM). Furthermore, by balance of the peptides' lipophilicity and biological activity, inhibitory activity against NNMT in cell-based assay was successfully achieved (peptide 26: cell-based IC50 = 770 nM). These findings illuminate the potential of cyclic peptides as a relatively new drug discovery modality even for intracellular targets.

Structure-Based Approaches to Improving Selectivity through Utilizing Explicit Water Molecules: Discovery of Selective ß-Secretase (BACE1) Inhibitors over BACE2.

BACE1 is an attractive target for disease-modifying treatment of Alzheimer's disease. BACE2, having high homology around the catalytic site, poses a critical challenge to identifying selective BACE1 inhibitors. Recent evidence indicated that BACE2 has various roles in peripheral tissues and the brain, and therefore, the chronic use of nonselective inhibitors may cause side effects derived from BACE2 inhibition. Crystallographic analysis of the nonselective inhibitor verubecestat identified explicit water molecules with different levels of free energy in the S2' pocket. Structure-based design targeting them enabled the identification of propynyl oxazine 3 with improved selectivity. Further optimization efforts led to the discovery of compound 6 with high selectivity. The cocrystal structures of 7, a close analogue of 6, bound to BACE1 and BACE2 confirmed that one of the explicit water molecules is displaced by the propynyl group, suggesting that the difference in the relative water displacement cost may contribute to the improved selectivity.

Balancing potency and basicity by incorporating fluoropyridine moieties: Discovery of a 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitor that affords robust and sustained central Aß reduction.

ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the treatment of Alzheimer's disease (AD). In this report, we describe the discovery of BACE1 inhibitors with a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine rings, which showed increased biochemical and cellular potency, along with reduced basicity on the amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained cerebrospinal fluid (CSF) Aß reduction in dog. The crystal structure of compound 11 bound to BACE1 confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.

Discovery of Atabecestat (JNJ-54861911): A Thiazine-Based ß-Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor Advanced to the Phase 2b/3 EARLY Clinical Trial.

Accumulation of amyloid ß peptides (Aß) is thought to be one of the causal factors of Alzheimer's disease (AD). The aspartyl protease ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting protease for Aß production, and therefore, BACE1 inhibition is a promising therapeutic approach for the treatment of AD. Starting with a dihydro-1,3-thiazine-based lead, Compound J, we discovered atabecestat 1 (JNJ-54861911) as a centrally efficacious BACE1 inhibitor that was advanced into the EARLY Phase 2b/3 clinical trial for the treatment of preclinical AD patients. Compound 1 demonstrated robust and dose-dependent Aß reduction and showed sufficient safety margins in preclinical models. The potential of reactive metabolite formation was evaluated in a covalent binding study to assess its irreversible binding to human hepatocytes. Unfortunately, the EARLY trial was discontinued due to significant elevation of liver enzymes, and subsequent analysis of the clinical outcomes showed dose-related cognitive worsening.

A case of congenital herpes simplex virus infection diagnosed at 8 months of age.

We present the case of an 8-month-old boy with the repeated recurrence of vesicles from the time of birth and who subsequently manifested psychomotor developmental delay. We retrospectively diagnosed the patient with congenital herpes simplex virus (HSV) infection. Computed tomography showed multiple calcifications in the periventricular white matter and thalami. The bilateral deep white matter showed an abnormally high signal intensity on T2-weighted magnetic resonance imaging. The patient required consecutive, suppressive therapy with valacyclovir to prevent the repeated recurrence of vesicles. This case presented a milder phenotype of congenital HSV infection in comparison to previous reports, and highlights the importance of the careful examination for this disease when neonates present with skin lesions.

Non-invasive discrimination of acute focal bacterial nephritis with pyelonephritis.

BACKGROUND: The appropriate antimicrobial treatment period for febrile urinary tract infection (UTI) can be changed, depending on whether the patient has acute focal bacterial nephritis (AFBN). The aim of this study was to clarify the characteristics of AFBN compared with those of acute pyelonephritis (APN) and establish a strategy to detect AFBN. METHODS: A total of 77 patients diagnosed with febrile UTI were enrolled. They were divided into APN (n = 64) and AFBN groups (n = 13). The clinical data and other laboratory biomarkers were retrospectively analyzed. RESULTS: The time required for fever resolution after antimicrobial treatment was significantly longer in the AFBN group than in the APN group (2.77 days vs 1.11 days, respectively, P < 0.001). Also, the time to disappearance of pyuria after antimicrobial treatment was longer in the AFBN group than in the APN group (6.22 days vs 2.32 days, respectively, P = 0.001). Fever lasting >1.75 days after antimicrobial treatment had a sensitivity of 92% and specificity of 79% for the detection of AFBN, while pyuria disappearance after 4 days had a sensitivity of 88% and specificity of 85%. When patients fulfilled both cut-offs, the sensitivity and specificity were 89% and 97%. CONCLUSION: Acute focal bacterial nephritis was associated with fever of significantly longer duration after antimicrobial treatment, and it took a longer time for pyuria to disappear. Children with febrile UTI should be evaluated for AFBN if the fever persists ≥48 h after the initiation of antimicrobial treatment and if pyuria lasts for 4 days.

Discovery of a potent orally bioavailable retinoic acid receptor-related orphan receptor-gamma-t (RORγt) inhibitor, S18-000003.

The retinoic acid receptor-related orphan receptor-gamma-t (RORγt) is the master transcription factor responsible for regulating the development and function of T-helper 17 (Th17) cells, which are related to the pathology of several autoimmune disorders. Therefore, RORγt is an attractive drug target for such Th17-mediated autoimmune diseases. A structure-activity relationship (SAR) study of lead compound 1 yielded a novel series of RORγt inhibitors, represented by compound 6. Detailed SAR optimization, informed by X-ray cocrystal structure analysis, led to the discovery of a potent orally bioavailable RORγt inhibitor 25, which inhibited IL-17 production in the skin of IL-23-treated mice by oral administration.

RT-qPCR analysis of the tumor antigens TOMM34 and RNF43 in samples extracted from paraffin-embedded specimens of colorectal cancer.

Comprehensive genetic analysis of colorectal malignant tumors by microarrays has identified translocase of the outer mitochondrial membrane 34 (TOMM34) and ring finger protein 43 (RNF43) as highly expressed oncogenes in malignant colorectal tumors. Vaccine therapy using cancer peptides synthesized using the amino acid sequences of tumor antigens is currently undergoing clinical trials. Since it is important to perform vaccine therapy based on the oncogene expression levels in individual tumors, analysis of tumor antigen expression is necessary for this therapy. However, the quality of the messenger RNA extracted from formalin-fixed and paraffin-embedded specimens is generally considered insufficient for gene quantification. The present study examined whether it could be possible to quantify the expression of TOMM34 and RNF43 in colorectal cancer and liver metastasis samples prepared from paraffin blocks. The formalin-fixed and paraffin-embedded specimens were sliced for slides and the colorectal cancer and normal mucosal tissues were obtained from the slides. Total RNA was extracted from the tissue samples, and quantitative polymerase chain reaction (qPCR) was performed using the Universal ProbeLibrary as a PCR probe. Quantification of TOMM34 and RNF43 gene expression in several-year-old paraffin-embedded colorectal cancer specimens was possible by qPCR using the Universal ProbeLibrary. qPCR revealed that TOMM34 expression was elevated in 78.9% (15 of 19 cases) of the primary tumors and in 73.7% (14 of 19 cases) of the liver metastasis samples. RNF43 expression was elevated in 63.2% (12 of 19 cases) of the primary tumors and in 73.7% (14 of 19 cases) of the liver metastasis samples.

Serial sonographic findings during progression from acute pyelonephritis to renal abscess: a rare case report.

Renal abscess, accumulation of infective fluid in the kidney, is a rare pathology. Currently, no reports of the serial imaging changes of acute pyelonephritis (APN) progressing to renal abscess exist. We report clinical and serial sonographic findings of a patient with hyper-immunoglobulin E syndrome, a primary immunodeficiency, who developed APN that progressed to renal abscess. Renal ultrasonography revealed that echogenicity of infectious lesions dramatically changed from isoechoic to hyperechoic and to hypoechoic during progression. These findings are useful for differential diagnosis of APN, acute focal bacterial nephritis, and renal abscess.

Focal frontal epileptiform discharges in a patient with eyelid myoclonia and absence seizures.

Eyelid myoclonia with absences is classified as a unique type of generalized seizure. Its pathogenesis is proposed to involve the functional abnormalities in cortical-subcortical networks. Here, we describe the case of a 7-year-old boy who had eyelid myoclonia with absences, along with focal motor seizures. Video-EEG monitoring demonstrated eyelid myoclonia associated with 4- to 5-Hz generalized polyspike-waves preceded by focal frontal discharges. Interictal EEG showed focal epileptiform discharges over the frontal regions. Our case suggests an important role of the frontal lobe in the generation of eyelid myoclonia with absences.

Electroclinical features of epileptic encephalopathy caused by SCN8A mutation.

Voltage-gated sodium channel Nav 1.6, encoded by the gene SCN8A, plays a crucial role in controlling neuronal excitability. SCN8A mutations that cause increased channel activity are associated with seizures. We describe a patient with epileptic encephalopathy caused by de novo SCN8A mutation (c.5614C>T, p.Arg1872Trp). Seizures began 10 days after birth at which time brain magnetic resonance imaging (MRI) and electroencephalography (EEG) were normal. Seizure recurrence increased with age, leading to the development of frequent status epilepticus from 1 year of age. Seizure type included generalized tonic seizures and focal motor seizures. EEG first showed focal epileptic activity at the age of 4 months, and thereafter showed multifocal spikes. Serial MRI demonstrated brain atrophy, which appeared to progress with seizure aggravation. Clinical features that may give a clue to the diagnosis include normal EEG despite frequent seizures in early infancy and an increase in epileptic activity that occurs with aging.

Human herpesvirus-6 infection-associated acute encephalopathy without skin rash.

BACKGROUND: Human herpesvirus-6 (HHV-6) is the etiological agent of exanthema subitum-associated encephalopathy, which usually occurs in children younger than 3 years. Brain imaging shows various abnormalities. PATIENT: A previously healthy 4-year-old girl developed acute encephalopathy with clinical features consisting of fever, repetitive seizures, and a disturbance of consciousness. The patient did not show skin rash suggestive of exanthema subitum during the course of her illness. The primary HHV-6 infection was diagnosed based on the absence of IgG against HHV-6 and identification of the virus DNA in the acute phase serum and a significant increase of the anti-HHV-6 IgG titers in the convalescent phase sera. Diffusion-weighted images showed transient high signal intensity in the bilateral periventricular white matter and splenium of the corpus callosum and in the gray matter structures such as the bilateral basal ganglia and thalami. Upon therapy with steroid and γ-globulin, the patient recovered without any neurological deficits. CONCLUSION: Primary HHV-6 infection can cause acute encephalopathy without exanthema subitum. The etiological diagnosis is possible only by examining the blood and cerebrospinal fluid, when the patient shows no skin rash. This condition should be included in the differential diagnosis of acute encephalopathy even in patients older than 3 years.

Crystallization and preliminary X-ray crystallographic analysis of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA.

Dihydrouridine synthase (Dus) is responsible for catalyzing dihydrouridine formation in RNA by the reduction of uridine. To elucidate its RNA-recognition mechanism, Dus from Thermus thermophilus (TthDus) and its complex with tRNA were crystallized. Diffraction data sets were collected from crystals of native and selenomethionine-substituted TthDus to resolutions of 1.70 and 2.30 Å, respectively. These crystals belonged to space group P1. Preliminary X-ray crystallographic analysis showed that two molecules of TthDus were contained in an asymmetric unit. In addition, diffraction data were collected to 3.51 Šresolution from a crystal of selenomethionine-substituted TthDus in complex with tRNA, which belonged to space group P4(1)2(1)2. Preliminary structural analysis showed that the asymmetric unit contained two TthDus-tRNA complexes.

Early induction of interleukin-5 and peripheral eosinophilia in acute pneumonia in Japanese children infected by pandemic 2009 influenza A in the Tokyo area.

A novel influenza A (2009 H1N1) virus has led to a worldwide pandemic. A significant number of patients with pneumonia have been reported, although its pathogenesis remains to be elucidated. To determine its pathogenesis, we evaluated serum interleukin (IL)-5 and peripheral eosinophil counts in patients with acute pneumonia caused by the 2009 H1N1 virus. During the period from October to December 2009, 40 patients with laboratory-confirmed 2009 H1N1 pneumonia were under investigation. Their mean age at presentation was 6.8 years. The most characteristic finding was the early development of hypoxemic respiratory distress in the first 24 hr after the onset of fever. Bronchial mucous plugs included eosinophils in addition to neutrophils, even in patients without allergies. Serum IL-5 levels were elevated in 20 out of 24 patients (83%) whose samples were obtained in the first 24 hr after the onset of fever (26.5 ± 20.1 pg/mL), independent of the presence of underlying allergies. In contrast, induction of IL-5 was not documented in sera from eight patients with laboratory-confirmed 2009 H1N1 virus who developed neurological complications, but without lower respiratory infection (2.1 ± 0.7 pg/mL, P < 0.001 vs acute pneumonia). Peripheral eosinophilia was characteristic in acute pneumonia, but not in patients without a lower respiratory infection. There was a marked difference in the induction of IL-5 in 2009 H1N1 patients who developed acute pneumonia, compared with those without a lower respiratory infection. IL-5 may play a role in the early phase of acute pneumonia caused by the 2009 H1N1 virus in Japanese children.

Virtual CO2 MDCT pancreatography: a new feasible technique for minimally invasive pancreatectomy in intraductal papillary mucinous neoplasms.

BACKGROUND/AIMS: Less invasive pancreatic head resection, such as duodenum-preserving pancreatic head resection (DPPHR) has been introduced for the treatment of pancreatoduodenal lesions, especially for benign conditions, for reducing surgical stress and maintaining exocrine and endocrine function of the residual pancreas in consideration of postoperative quality of life (QOL). METHODOLOGY: We investigated the feasibility of a new technique employing three-dimensional (3D) virtual pancreatography using multi-detector CT (MDCT) with carbon dioxide (CO2) gas as a negative contrast agent for detection of intraductal papillary mucinous neoplasm (IPMN) of the pancreas requiring minimally invasive surgery. Branch IPMN is subjected in this study. RESULTS: Contrast-enhanced MDCT scan of the abdomen diagnosed 4- to 20-mm multilocular septated cysts in the head-uncinate process of the pancreas. Endoscopic retrograde pancreatography (ERP) showed multiple cystic lesions in the head-uncinate process with mild dilatation in the remaining pancreatic duct. For localizing diagnosis of these small and multiple pancreatic cysts, we placed an endoscopic pancreatic stent (EPS), and MDCT with injection of CO2 via EPS was examined for the virtual CO2 pancreatography, consisting of OsiriX software system employing 3D virtual anatomic reconstruction with CO2 gas as a negative contrast agent. Virtual CO2 MDCT pancreatography demonstrated that all cystic lesions of the pancreas were contained within the area of the head-uncinate process of the pancreas. We performed DPPHR, and surgical margin of the patient's remnant pancreas was determined as non-malignant by intraoperative histology. There was no residual pancreatic cyst and tumor after surgery. The resected tumor was diagnosed as branch duct type intraductal papillary mucinous adenocarcinoma. According to our minimally invasive DPPHR obtained by virtual CO2 pancreatography, the pancreatic endocrine and exocrine functions of this patient were maintained at almost the same levels as those in his preoperative status. With respect to preservation of the endocrine and exocrine functions of the pancreas, DPPHR is a highly effective surgical procedure due to limited surgical resection. CONCLUSIONS: Our new technique of virtual CO2 MDCT pancreatography is a feasible procedure for preservation of the remnant pancreatic function. This is the first report of virtual CO2 pancreatography providing minimally invasive pancreatic surgery.