Evolutionary Design of Self-Templated Supramolecular Fibrils Using M13 Bacteriophage for Tissue Engineering.

Biomaterials in nature form hierarchical structures and functions across various length scales through binding and assembly processes. Inspired by nature, we developed hierarchically organized tissue engineering materials through evolutionary screening and self-templating assembly. Leveraging the M13 bacteriophage (phage), we employed an evolutionary selection process against hydroxyapatite (HA) to isolate HA-binding phage (HAPh). The newly discovered phage exhibits a bimodal length, comprising 950 nm and 240 nm, where the synergistic effect of these dual lengths promotes the formation of supramolecular fibrils with periodic banded structures. The assembled HAPh fibrils show the capability of HA mineralization and the directional growth of osteoblast cells. When applied to a dentin surface, it induces the regeneration of dentin-like tissue structures, showcasing its potential applications as a scaffold in tissue engineering. The integration of evolutionary screening and self-templating assembly holds promise for the future development of hierarchically organized tissue engineering materials.

Determination of Leuprolide-Fatty Acid Conjugate in Rat Plasma Using LC-MS/MS and Its Pharmacokinetics after Subcutaneous Administration in Rats.

Leuprolide is a synthetic nonapeptide drug (pyroGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt) that acts as a gonadotropin-releasing hormone agonist. The continuous administration of therapeutic doses of leuprolide inhibits gonadotropin secretion, which is used in androgen-deprivation therapy for the treatment of advanced prostate cancer, central precocious puberty, endometriosis, uterine fibroids, and other sex-hormone-related conditions. To improve the pharmacokinetic properties of peptide drugs, a fatty acid was conjugated with leuprolide for long-term action. In this study, we developed a simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of leuprolide and leuprolide-oleic acid conjugate (LOC) levels. The developed method was validated in terms of linearity, precision, accuracy, recovery, matrix effect, and stability according to the US Food and Drug Administration guidelines, and the parameters were within acceptable limits. Subsequently, the pharmacokinetics of leuprolide and LOCs were evaluated. In vivo rat subcutaneous studies revealed that conjugation with fatty acids significantly altered the pharmacokinetics of leuprolide. After the subcutaneous administration of fatty-acid-conjugated leuprolide, the mean absorption time and half-life were prolonged. To the best of our knowledge, this is the first study showing the effects of fatty acid conjugates on the pharmacokinetics of leuprolide using a newly developed UPLC-MS/MS method for the simultaneous quantification of leuprolide and LOCs.

Mitoxantrone-Loaded PEGylated Gold Nanocomplexes for Cancer Therapy.

Gold nanoparticles (AuNPs) have been widely used as drug delivery carriers for cancer targeting and therapy. In this study, we developed mitoxantrone (MX)-loaded poly(ethylene glycol)-modified AuNPs complexes (AuNPs-PEG-MX) and evaluated its physicochemical properties compared to AuNPs, free MX, and MX-loaded AuNPs (AuNPs-MX). The results of surface plasmon resonance (SPR) measurement provided corresponded characteristics of free MX and AuNP groups, which determined by electrophoretic light scattering (ELS) method. The hydrodynamic size of AuNPs-PEG-MX was lower than that of AuNPs-MX. Furthermore, loading efficiency of AuNPs-PEG-MX was 1.9-fold increased than AuNPs-MX. In addition, AuNPs-PEG-MX showed similar cytotoxicity compared to AuNPs-MX group in HeLa cells with enhanced drug release. Conclusively, AuNPs-PEG-MX could be applied for in vivo cancer therapy via passive targeting based on the enhanced permeability and retention effect after intravenous injection.

Elastin-Based Rubber-Like Hydrogels.

We developed rubber-like elastomeric materials using a natural elastin derived sequence and genetic engineering to create precisely defined elastin-like polypeptides. The coiled elastin-like polypeptide chains, which behave like entropic springs, were cross-linked using an end-to-end tethering scheme to synthesize simple hydrogels with excellent extensibility and reversibility. Our hydrogels extend to strains as high as 1500% and remain highly resilient with elastic recovery as high as 94% even at 600% strain, significantly exceeding any other protein-based hydrogel. These materials are valuable as elastomeric hydrogels for designing extremely robust scaffolds useful for tissue engineering.

Self-Healing Elastin-Bioglass Hydrogels.

Tailorable hydrogels that are mechanically robust, injectable, and self-healable, are useful for many biomedical applications including tissue repair and drug delivery. Here we use biological and chemical engineering approaches to develop a novel in situ forming organic/inorganic composite hydrogel with dynamic aldimine cross-links using elastin-like polypeptides (ELP) and bioglass (BG). The resulting ELP/BG biocomposites exhibit tunable gelling behavior and mechanical characteristics in a composition and concentration dependent manner. We also demonstrate self-healing in the ELP/BG hydrogels by successfully reattaching severed pieces as well as through rheology. In addition, we show the strength of genetic engineering to easily customize ELP by fusing cell-stimulating "RGD" peptide motifs. We showed that the resulting composite materials are cytocompatible as they support the cellular growth and attachment. Our robust in situ forming ELP/BG composite hydrogels will be useful as injectable scaffolds for delivering cell and drug molecules to promote soft tissue regeneration in the future.

Selective and Sensitive Sensing of Flame Retardant Chemicals Through Phage Display Discovered Recognition Peptide.

We report a highly selective and sensitive biosensor for the detection of an environmentally toxic molecule, decabrominated diphenyl ether (DBDE), one of the most common congeners of the polybrominated frame retardants (polybrominated diphenyl ether (PBDE)), using newly discovered DBDE peptide receptors integrated with carbon nanotube field-effect transistors (CNT-FET). The specific DBDE peptide receptor was identified using a high-throughput screening process of phage library display. The resulting binding peptide carries an interesting consensus binding pocket with two Trp-His/Asn-Trp repeats, which binds to the DBDE in a multivalent manner. We integrated the novel DBDE binding peptide onto the CNT-FET using polydiacetylene coating materials linked through cysteine-maleimide click chemistry. The resulting biosensor could detect the desired DBDE selectively with a 1 fM detection limit. Our combined approaches of selective receptor discovery, material nanocoating through click chemistry, and integration onto a sensitive CNT-FET electronic sensor for desired target chemicals will pave the way toward the rapid development of portable and easy-to-use biosensors for desired chemicals to protect our health and environment.

Biomimetic Self-Templated Hierarchical Structures of Collagen-Like Peptide Amphiphiles.

Developing hierarchically structured biomaterials with tunable chemical and physical properties like those found in nature is critically important to regenerative medicine and studies on tissue morphogenesis. Despite advances in materials synthesis and assembly processes, our ability to control hierarchical assembly using fibrillar biomolecules remains limited. Here, we developed a bioinspired approach to create collagen-like materials through directed evolutionary screening and directed self-assembly. We first synthesized peptide amphiphiles by coupling phage display-identified collagen-like peptides to long-chain fatty acids. We then assembled the amphiphiles into diverse, hierarchically organized, nanofibrous structures using directed self-assembly based on liquid crystal flow and its controlled deposition. The resulting structures sustained and directed the growth of bone cells and hydroxyapatite biominerals. We believe these self-assembling collagen-like amphiphiles could prove useful in the structural design of tissue regenerating materials.

Cyclic RGD peptide incorporation on phage major coat proteins for improved internalization by HeLa cells.

Delivering therapeutic materials or imaging reagents into specific tumor tissues is critically important for development of novel cancer therapeutics and diagnostics. Genetically engineered phages possess promising structural features to develop cancer therapeutic materials. For cancer targeting purposes, we developed a novel engineered phage that expressed cyclic RGD (cRGD) peptides on the pVIII major coat protein using recombinant DNA technology. Using a type 88 phage engineering approach, which inserts a new gene to express additional major coat protein in the noncoding region of the phage genome, we incorporated an additional pVIII major coat protein with relatively bulky cRGD and assembled heterogeneous major coat proteins on the F88.4 phage surfaces. With IPTG control, we could tune different numbers of cRGD peptide displayed on the phage particles up to 140 copies. The resulting phage with cRGD on the recombinant pVIII protein exhibited enhanced internalization efficiency into HeLa cells in a ligand density and conformational structure dependent manner when comparing with the M13 phages modified with either linear RGD on pVIII or cRGD on pIII. Our cRGD peptide engineered phage could be useful for cancer therapy or diagnostic purposes after further modifying the phage with drug molecules or contrast reagents in the future.

Recent bioanalytical methods for quantification of third-generation cephalosporins using HPLC and LC-MS(/MS) and their applications in pharmacokinetic studies.

Third-generation cephalosporins are semi-synthetic antibiotics with enhanced activity against Gram-negative organisms. The cephalosporins in biological samples are mostly determined using high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS). In recent years, numerous bioanalytical methods have been developed to improve the sensitivity and specificity of cephalosporin quantification using the powerful LC-MS/MS systems that are common in research laboratories. This review article presents recently developed bioanalytical methods by HPLC or LC-MS(/MS) for 12 third-generation cephalosporins, including five oral third-generation cephalosporins, and further discusses their application in pharmacokinetic studies of animals and humans.

Transport of gemifloxacin, a 4th generation quinolone antibiotic, in the Caco-2 and engineered MDCKII cells, and potential involvement of efflux transporters in the intestinal absorption of the drug.

The oral (po) bioavailability of gemifloxacin mesylate in rats and its possible association with efflux transporters was investigated. The apparent permeabilities (Papp) of gemifloxacin across the Caco-2 cell monolayer were 1.20 ± 0.09 × 10(-5) cm/s for apical to basal (absorptive) transport, and 2.13 ± 0.6 × 10(-5) cm/s for basal to apical (secretory) transport for a 5-500 µM concentration range, suggesting the involvement of a carrier-mediated efflux in the secretory transport. The secretory transport in Caco-2 cells was significantly decreased by MRP2 (MK571) and BCRP (Ko143) inhibitors. The secretory transport was distinct in MDCKII/P-gp, MDCKII/MRP2 and MDCKII/BCRP cells, and the affinity was highest for MRP2, followed by BCRP and P-gp. The efflux was significantly decreased by verapamil and Ko143, but not significantly by MK571. The comparative po bioavailability in rats was increased by the preadministration of Ko143 (four-fold), MK571 (two-fold) and verapamil (two-fold). Efflux transporters appeared to significantly limit the bioavailability of gemifloxacin in rats, suggesting their possible contribution to the low bioavailability of the drug in the human (70%).

Determination of cefdinir levels in rat plasma and urine by high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics after oral and intravenous administration of cefdinir.

A selective and sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination of cefdinir in rat plasma and urine. Following a simple protein precipitation using methanol, chromatographic separation was achieved with a run time of 10 min using a Synergi 4 µ polar-RP 80A column (150 × 2.0 mm, 4 µm) with a mobile phase consisting of 0.1% formic acid in water and methanol (65:35, v/v) at a flow rate of 0.2 mL/min. The protonated precursor and product ion transitions for cefdinir (m/z 396.1 → 227.2) and cefadroxil, an internal standard (m/z 364.2 → 208.0) were monitored in the multiple reaction monitoring in positive ion mode. The calibration curves for plasma and urine were linear over the concentration range 10-10,000 ng/mL. The lower limit of quantification was 10 ng/mL. All accuracy values were between 95.1 and 113.0% and the intra- and inter-day precisions were <13.0% relative standard deviation. The stability under various conditions in rat plasma and urine was also found to be acceptable at three concentrations. The developed method was applied successfully to the pharmacokinetic study of cefdinir after oral and intravenous administration.

An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.

The Biopharmaceutics Classification System (BCS) was developed to facilitate estimation of the in vivo pharmacokinetic performance of drugs from human intestinal permeability and solubility. However, the measurement of human in vivo intestinal permeability, unlike that of solubility, is problematic and inefficient. Thus, rat in vitro intestinal permeability results obtained via the Ussing chamber technique are often used instead. However, these data could be unreliable due to difficulty in maintaining the viability of the dissected intestinal membrane in the Ussing chamber. Therefore, a more efficient method to obtain a reliable in vitro permeability is mandatory. Here, we propose a new approach by introducing a novel factor called the permeability ratio (PR). Basically, PR is a rat in vitro intestinal permeability obtained from the Ussing chamber, which is then corrected by the permeability of lucifer yellow, a paracellular permeability marker. To prove the validity of the method, 12 model drugs representing different BCS classes were tested, and the correlation with human in vivo intestinal permeability was high. More importantly, the new method perfectly classified all 12 model drugs. The results indicate that PR is a reliable factor with high correlation to human in vivo intestinal permeability, which can further be used to accurately predict the BCS classification.

Coiled-coil structure mediated inhibition of the cytotoxic huntingtin amyloid fibrils by an IP3 receptor fragment.

Disruption of cellular homeostasis by the aggregation of polyglutamine (polyQ) in the huntingtin protein (Htt) leads Huntington's disease (HD). Effective drugs for treating HD have not been developed, as the molecular mechanism underlying HD pathogenesis remains unclear. To develop strategies for inhibiting HD pathogenesis, the intermolecular interaction of Htt with IP3 receptor 1 (IP3R1) was investigated. Peptide (termed ICT60) corresponding to a coiled-coil motif in the C-terminus of IP3R1 was designed. Several biophysical approaches revealed the strong and specific binding of ICT60 to the N-terminal part of HttEx1. ICT60 inhibited not only amyloid formation by HttEx1, but also the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1. The importance of coiled-coil structure was verified by charge-manipulated variants. The coiled-coil structures of ICT60-KK and -EE were partially and largely disrupted, respectively. ICT60 wild-type and -KK inhibited amyloid formation by HttEx1-46Q, whereas ICT60-EE did not block amyloidogenesis. Similarly, the cytotoxicity and cell-penetration ability of the amyloid fibrils of HttEx1-46Q were efficiently inhibited by ICT60 wild-type and ICT60-KK, but not by ICT60-EE. We propose a mechanical model explaining how an IP3 receptor-inspired molecule can modulate cytotoxic amyloid formation by Htt, providing a molecular basis for developing therapeutics to treat HD.

Development of Squalene-Based Oil-in-Water Emulsion Adjuvants Using a Self-Emulsifying Drug Delivery System for Enhanced Antigen-Specific Antibody Titers.

Introduction: A recombinant protein cannot induce sufficient immune response by itself. Various substances, including cytokine and mineral, have been used as adjuvants to enhance the immunogenicity and efficacy of vaccines; however, most of them induce excessive immune responses or exhibit cytotoxicity. In this study, a self-emulsifying drug delivery system (SEDDS), an isotropic mixture of oil, surfactant, and solvent, was designed for oil-in-water emulsions as a non-toxic adjuvant to increase immune response to antigens. Methods: Squalene-based oil-in-water emulsions were prepared by SEDDS to assess its value as an adjuvant. Fifteen emulsions (F1-F15) were prepared by stirring two types of surfactants (Span® 85 and Kolliphor® RH40), and squalene and carboxymethyl cellulose (CMC) were added at different ratios. The physical properties and viscosity of the 15 emulsions were evaluated by measuring droplet size, zeta potential, and polydispersity index. The toxic effect of emulsions was assessed by acute toxicity test in mice. Mice were immunized twice with 1:1 mixtures of antigen and adjuvant (15 emulsions, phosphate-buffered saline, and commercial alum-based adjuvant). Antigen-specific antibody titers from immunized mice serum were measured by an indirect enzyme-linked immunosorbent assay. Results: All emulsions exhibited droplet sizes ranging from 322 to 812 nm and maintained zeta potential values between -30 mV to -10 mV for 4 weeks, indicating good physical stability as a vaccine adjuvant. Additionally, all emulsions were non-toxic, and they induced humoral immunity at a similar level compared to commercial alum-based adjuvant in the first immunization. However, 12% squalene-based oil-in-water emulsion containing 0.5% of ultra-high viscosity CMC (F15) showed significantly higher immune response than a commercial adjuvant in the second immunization. Conclusion: Squalene-based oil-in-water emulsions could be conveniently prepared using SEDDS technique and are non-toxic and stable at room temperature storage. Moreover, squalene-based oil-in-water emulsions show enhanced immune induction with antigen; hence, they can possibly be used as effective adjuvants.

Dataset for hierarchical tetramodal-porous architecture of zinc oxide nanoparticles microfluidically synthesized via dual-step nanofabrication.

Zinc oxide (ZnO) nanoparticles (NPs) have been applied as high-performance intelligent materials to create a hierarchical multimodal-porous architectures for application in biomedical research fields [1]. They were microfluidically synthesized via dual-step nanofabrication compared to the conventional particles including ZnO NPs synthesized at single-pot macroscale, nanosized ZnO, and hybrid ZnO. The physicochemical properties were characterized, including morphology, particle size distribution, atomic composition, crystallinity, purity, reactant viscosity, surface charge, photocatalysis, photoluminescence, and porosity. A hierarchical multimodal-porous three-dimensional (3D) architecture of ZnO NPs was generated and optimized on the solid plate substrate of cellulose paper sheet after solvent evaporation. The dataset provides the nanomaterial design and architecture generation of ZnO NPs, explaining multi-physics phenomena in association with performance optimization processes.

Hydroxyl Group-Targeted Conjugate and Its Self-Assembled Nanoparticle of Peptide Drug: Effect of Degree of Saturation of Fatty Acids and Modification of Physicochemical Properties.

Purpose: To conjugate different degree of saturation of C18 fatty acids (stearic acid, oleic acid, and linoleic acid) with the hydroxyl groups of leuprolide acetate (LEU acetate) and to investigate the controlled release and enhanced permeability through self-assembled nanoparticles (L18FNs). Methods: Yamaguchi esterification with benzoyl chloride and DMAP (4-Dimethylaminopyridine) allowed the conjugation of the fatty acid to the hydroxyl group of LEU. The three conjugates were then designated as stearic acid-conjugated LEU, LSC, oleic acid-conjugated LEU, LOC, and linoleic acid-conjugated LEU, LLC, respectively. The conjugates (L18FCs) were purified using preparative HPLC (Prep-HPLC) and identified through various instrumental analyses. Results: The zeta potential, particle size, and morphology of each L18FNs were evaluated. In the case of LSNs, the zeta potential value was relatively low and the particle size was larger than LONs and LLNs owing to the higher hydrophobicity of saturated fatty chain, while the LLNs showed a higher zeta potential and smaller particle size. In human plasma, LLC showed the fastest degradation rate with the highest accumulative drug release. The permeability of L18FNs was analyzed through the Franz diffusion cell experiment, confirming that the degree of saturation of fatty acids affects the permeability of LFNs. While the permeability of LSNs was not significantly enhanced due to higher particle size after nanonization, LONs and LLNs increased 1.56 and 1.85 times in permeation, respectively, compared to LEU. Conclusion: Utilization of different degree of saturation of fatty acids to conjugate a peptide drug could provide pharmaceutical versatility via self-assembly and modification of physicochemical properties.

Fluorescence detection of Zabofloxacin, a novel fluoroquinolone antibiotic, in plasma, bile, and urine by HPLC: the first oral and intravenous applications in a pharmacokinetic study in rats.

PURPOSE: To develop an HPLC method using fluorescence detection for the pharmacokinetic evaluation of levels of zabofloxacin, a novel broad spectrum fluoroquinolone antibiotic, in the plasma, bile and urine of rats. METHODS: A simple reversed-phase HPLC method using a C18 column with fluorescence detection was developed and validated for the simultaneous determination of zabofloxain and enrofloxacin as an internal standard. The plasma sample was treated with methanol for protein precipitation, and treatment of the bile and urine samples included deproteinization and extraction using chloroform. The applicability of the developed assay method to pharmacokinetic studies of zabofloxacin in rats was examined. Zabofloxacin was intravenously and orally administered to rats at a dose of 20 mg/kg. RESULTS: The limits of quantification (LOQ) was determined to be 50 ng/mL for the plasma with acceptable linearity ranging from 50 to 25,000 ng/mL (R>0.999), and 0.5 µg/mL for the bile and urine samples with acceptable linearity ranging from 0.5 to 100 µg/mL (R>0.999). The validation parameters for zabofloxacin were found to be acceptable according to FDA assay validation (2001). While zabofloxacin in plasma and urine has been stable in all tested handling conditions, it has been unstable in bile during freeze-thaw cycles for 24 h at room temperature. Following intravenous and oral administration of zabofloxacin to rats at a dose of 20 mg/kg, concentration was quantifiable in plasma for up to 8 h. The bioavailability of zabofloxacin was 27.7%, and it was excreted into bile and urine at about 8% each per oral administration. CONCLUSIONS: These observations suggest that a validated assay can be used in pharmacokinetic studies of zabofloxacin in small animals. Due to the limited stability of zabofloxcin in rat bile, freeze-thaw cycles or prolonged handling at room temperature is not recommended. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Antimicrobial Activity of Zinc Oxide Nano/Microparticles and Their Combinations against Pathogenic Microorganisms for Biomedical Applications: From Physicochemical Characteristics to Pharmacological Aspects.

Zinc oxide (ZnO) nano/microparticles (NPs/MPs) have been studied as antibiotics to enhance antimicrobial activity against pathogenic bacteria and viruses with or without antibiotic resistance. They have unique physicochemical characteristics that can affect biological and toxicological responses in microorganisms. Metal ion release, particle adsorption, and reactive oxygen species generation are the main mechanisms underlying their antimicrobial action. In this review, we describe the physicochemical characteristics of ZnO NPs/MPs related to biological and toxicological effects and discuss the recent findings of the antimicrobial activity of ZnO NPs/MPs and their combinations with other materials against pathogenic microorganisms. Current biomedical applications of ZnO NPs/MPs and combinations with other materials are also presented. This review will provide the better understanding of ZnO NPs/MPs as antibiotic alternatives and aid in further development of antibiotic agents for industrial and clinical applications.

Multiscale Metal Oxide Particles to Enhance Photocatalytic Antimicrobial Activity against Escherichia coli and M13 Bacteriophage under Dual Ultraviolet Irradiation.

Antimicrobial activity of multiscale metal oxide (MO) particles against Escherichia coli (E. coli) and M13 bacteriophage (phage) was investigated under dual ultraviolet (UV) irradiation. Zinc oxide (ZnO), magnesium oxide (MgO), cuprous oxide (Cu2O), and cupric oxide (CuO) were selected as photocatalytic antimicrobials in MO particles. Physicochemical properties including morphology, particle size/particle size distribution, atomic composition, crystallinity, and porosity were evaluated. Under UV-A and UV-C irradiation with differential UV-C intensities, the antimicrobial activity of MO particles was monitored in E. coli and phage. MO particles had nano-, micro- and nano- to microscale sizes with irregular shapes, composed of atoms as ratios of chemical formulae and presented crystallinity as pure materials. They had wide-range specific surface area levels of 0.40-46.34 m2/g. MO particles themselves showed antibacterial activity against E. coli, which was the highest among the ZnO particles. However, no viral inactivation by MO particles occurred in phage. Under dual UV irradiation, multiscale ZnO and CuO particles had superior antimicrobial activities against E. coli and phage, as mixtures of nano- and microparticles for enhanced photocatalytic antimicrobials. The results showed that the dual UV-multiscale MO particle hybrids exhibit enhanced antibiotic potentials. It can also be applied as a next-generation antibiotic tool in industrial and clinical fields.

Development of Non-Ethoxypropanoic Acid Type Cryptochrome Inhibitors with Circadian Molecular Clock-Enhancing Activity by Bioisosteric Replacement.

Circadian dysfunction is closely associated with an increased risk of various diseases. Considering that molecular clock machinery serves as an intrinsic time-keeping system underlying the circadian rhythm of biological processes, the modulation of the molecular clock machinery is an attractive therapeutic target with novel mechanisms of action. Based on the previous structure-activity relationship study of small molecule cryptochrome (CRY) inhibitors possessing an ethoxypropanoic acid moiety, non-ethoxypropanoic acid-type inhibitors have been developed by bioisosteric replacement. They were evaluated as potent and effective enhancers of E-box-mediated transcription, and, in particular, ester 5d and its hydrolysis product 2d exhibited desirable metabolic and pharmacokinetic profiles as promising drug candidates. Compound 2d directly bound to both CRY1 and 2 in surface plasmon resonance analyses, suggesting that the molecular target is CRY. Effects of compound 5d and 2d on suppressive action of CRY1 on CLOCK:BMAL1-activated E-box-LUC reporter activity revealed that both compounds inhibited the negative feedback actions of CRY on CLOCK:BMAL1. Most importantly, compounds 5d and 2d exhibited significant effects on molecular circadian rhythmicity to be considered circadian clock-enhancers, distinct from the previously developed CRY inhibitors possessing an ethoxypropanoic acid moiety.