Repositioning of Thiourea-Containing Drugs as Tyrosinase Inhibitors

Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Developing functional modulators of tyrosinase is important for therapeutic and cosmetic purposes. Given the abundance of thiourea moiety in known tyrosinase inhibitors, we studied other thiourea-containing drugs as potential tyrosinase inhibitors. The thiourea-containing drugs in clinical use were retrieved and tested for their ability to inhibit tyrosinase. We observed that methimazole, thiouracil, methylthiouracil, propylthiouracil, ambazone, and thioacetazone inhibited mushroom tyrosinase. Except for methimazole, there was limited information regarding the activity of other drugs against tyrosinase. Both thioacetazone and ambazone significantly inhibited tyrosinase, with IC₅₀ of 14 and 15 μM, respectively. Ambazone decreased melanin content without causing cellular toxicity at 20 μM in B16F10 cells. The activity of ambazone was stronger than that of kojic acid both in enzyme and melanin content assays. Kinetics of enzyme inhibition assigned the thiourea-containg drugs as non-competitive inhibitors. The complex models by docking simulation suggested that the intermolecular hydrogen bond via the nitrogen of thiourea and the contacts via thione were equally important for interacting with tyrosinase. These data were consistent with the results of enzyme assays with the analogues of thiourea.     

Antamanide Analogs as Potential Inhibitors of Tyrosinase

The tyrosinase enzyme, which catalyzes the hydroxylation of monophenols and the oxidation of o-diphenols, is typically involved in the synthesis of the dark product melanin starting from the amino acid tyrosine. Contributing to the browning of plant and fruit tissues and to the hyperpigmentation of the skin, leading to melasma or age spots, the research of possible tyrosinase inhibitors has attracted much interest in agri-food, cosmetic, and medicinal industries. In this study, we analyzed the capability of antamanide, a mushroom bioactive cyclic decapeptide, and some of its glycine derivatives, compared to that of pseudostellarin A, a known tyrosinase inhibitor, to hinder tyrosinase activity by using a spectrophotometric method. Additionally, computational docking studies were performed in order to elucidate the interactions occurring with the tyrosinase catalytic site. Our results show that antamanide did not exert any inhibitory activity. On the contrary, the three glycine derivatives AG9, AG6, and AOG9, which differ from each other by the position of a glycine that substitutes phenylalanine in the parent molecule, improving water solubility and flexibility, showed tyrosinase inhibition by spectrophotometric assays. Analytical data were confirmed by computational studies.     

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety

We previously reported (E)-β-phenyl-α,β-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the β-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,β-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC₅₀ = 18.09 ± 0.25 μM), 1h (IC₅₀ = 4.14 ± 0.10 μM), and 2a (IC₅₀ = 15.69 ± 0.40 μM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC₅₀ = 48.62 ± 3.38 μM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.     

An Updated Review of Tyrosinase Inhibitors

Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed.     

Discovery of a Class of Potent and Selective Non-competitive Sentrin-Specific Protease 1 Inhibitors

Sentrin-specific proteases (SENPs) are responsible for the maturation of small ubiquitin-like modifiers (SUMOs) and the deconjugation of SUMOs from their substrate proteins. Studies on prostate cancer revealed an overexpression of SENP1, which promotes prostate cancer progression as well as metastasis. Therefore, SENP1 has been identified as a novel drug target against prostate cancer. Herein, we report the discovery and biological evaluation of potent and selective SENP1 inhibitors. A structure-activity relationship (SAR) of the newly identified pyridone scaffold revealed allosteric inhibitors with very attractive in vitro ADMET properties regarding plasma binding and plasma stability for this challenging target. This study also emphasizes the importance of biochemical mode of inhibition studies for de novo designed inhibitors.     

Ultrasonic-Assisted Synthesis of Benzofuran Appended Oxadiazole Molecules as Tyrosinase Inhibitors: Mechanistic Approach through Enzyme Inhibition,Molecular Docking,Chemoinformatics, ADMET and Drug-Likeness Studies

Furan-oxadiazole structural hybrids belong to the most promising and biologically active classes of oxygen and nitrogen containing five member heterocycles which have expanded therapeutic scope and potential in the fields of pharmacology, medicinal chemistry and pharmaceutics. A novel series 5a–j of benzofuran-oxadiazole molecules incorporating S-alkylated amide linkage have been synthesized using ultrasonic irradiation and screened for bacterial tyrosinase inhibition activity. Most of the synthesized furan-oxadiazole structural motifs exhibited significant tyrosinase inhibition activity in the micromolar range, with one of the derivatives being more potent than the standard drug ascorbic acid. Among the tested compounds, the scaffold 5a displayed more tyrosinase inhibition efficacy IC₅₀ (11 ± 0.25 μM) than the ascorbic acid IC₅₀ (11.5 ± 0.1 μM). Compounds 5b, 5c and 5d efficiently inhibited bacterial tyrosinase with IC₅₀ values in the range of 12.4 ± 0.0–15.5 ± 0.0 μM. The 2-fluorophenylacetamide containing furan-oxadiazole compound 5a may be considered as a potential lead for tyrosinase inhibition with lesser side effects as a skin whitening and malignant melanoma anticancer agent.     

Structure-Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis

Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low-fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first-line chemotherapy, with the majority of reported inhibitors targeting protein-protein interactions (PPIs) of the Rev1 C-terminal domain (Rev1-CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1-CT PPIs with Rev1-interacting regions (RIRs) of TLS polymerases. To explore the structure-activity relationships for this scaffold, we developed a protocol for co-crystallization of compounds that target the RIR binding site on Rev1-CT with a triple Rev1-CT/Rev7^R124A/Rev3-RBM1 complex, and solved an X-ray crystal structure of Rev1-CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1-CT. We synthesized eight additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1-CT by microscale thermophoresis (MST). Several second-generation PAP derivatives showed an affinity for Rev1-CT that was improved by over an order of magnitude, thereby validating the structure-based assumptions that went into the compound design.     

Design,Synthesis, and Structure-Activity Relationship Study of Pyrazolones as Potent Inhibitors of Pancreatic Lipase

Pancreatic lipase (PL), a key target for the prevention and treatment of obesity, plays crucial roles in the hydrolysis and absorption of in dietary fat. In this study, a series of pyrazolones was synthesized, and their inhibitory effects against PL were assayed by using 4-methylumbelliferyl oleate (4-MUO) as optical substrate for PL. Comprehensive structure–activity relationship analysis of these pyrazolones led us to design and synthesize a novel compound P32 (5-(naphthalen-2-yl)-2-phenyl-4-(thiophen-2-ylmethyl)-2,4-dihydro-3H-pyrazol-3-one) as a potent mixed-competitive inhibitor of PL (IC₅₀=0.30 μM). In addition, P32 displayed some selectivity over other known serine hydrolases. A molecular docking study for P32 demonstrated that the inhibitory activity of P32 towards PL could be attributed to the π-π interactions of 2-naphthyl unit (R¹) and hydrophobic interactions of phenyl moiety (R³) with the active site of PL. Thus, P32 could serve as promising lead compound for the development of more efficacious and selective pyrazolones-type PL inhibitors for biomedical applications.     

Variations in IC50 Values with Purity of Mushroom Tyrosinase

The effects of various inhibitors on crude, commercial and partially purified commercial mushroom tyrosinase were examined by comparing IC₅₀ values. Kojic acid, salicylhydroxamic acid, tropolone, methimazole, and ammonium tetrathiomolybdate had relatively similar IC₅₀ values for the crude, commercial and partially purified enzyme. 4-Hexylresorcinol seemed to have a somewhat higher IC₅₀ value using crude extracts, compared to commercial or purified tyrosinase. Some inhibitors (NaCl, esculetin, biphenol, phloridzin) showed variations in IC₅₀ values between the enzyme samples. In contrast, hydroquinone, lysozyme, Zn²⁺, and anisaldehyde showed little or no inhibition in concentration ranges reported to be effective inhibitors. Organic solvents (DMSO and ethanol) had IC₅₀ values that were similar for some of the tyrosinase samples. Depending of the source of tyrosinase and choice of inhibitor, variations in IC₅₀ values were observed.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

Strategies To Design Selective Histone Deacetylase Inhibitors

This review classifies drug-design strategies successfully implemented in the development of histone deacetylase (HDAC) inhibitors, which have many applications including cancer treatment. Our focus is on especially demanded selective HDAC inhibitors and their structure-activity relationships in relation to corresponding protein structures. The main part of the paper is divided into six subsections each narrating how optimization of one of six structural features can influence inhibitor selectivity. It starts with the impact of the zinc binding group on selectivity, continues with the optimization of the linker placed in the substrate binding tunnel as well as the adjustment of the cap group interacting with the surface of the protein, and ends with the addition of groups targeting class-specific sub-pockets: the side-pocket-, lower-pocket- and foot-pocket-targeting groups. The review is rounded off with a conclusion and an outlook on the future of HDAC inhibitor design.     

Homarine Alkyl Ester Derivatives as Promising Acetylcholinesterase Inhibitors

Reversible acetylcholinesterase (AChE) inhibitors are key therapeutic tools to modulate the cholinergic connectivity compromised in several degenerative pathologies. In this work, four alkyl esters of homarine were synthesized and screened by using Electrophorus electricus AChE and rat brain AChE-rich fraction. Results showed that all homarine alkyl esters are able to inhibit AChE by a competitive inhibition mode. The effectiveness of AChE inhibition increases with the alkyl side chain length of the homarine esters, being HO−C₁₆ (IC₅₀=7.57±3.32 μM and K_i=18.96±2.28 μM) the most potent inhibitor. The fluorescence quenching studies confirmed that HO−C₁₆ is the compound with higher selectivity and affinity for the tryptophan residues in the catalytic active site of AChE. Preliminary cell viability studies showed that homarine esters display no toxicity for human neuronal SH-SY5Y cells. Thus, the long-chain homarine esters emerge as new anti-cholinesterase agents, with potential to be considered for therapeutic applications development.     

Development of Novel Amides as Noncovalent Inhibitors of Immunoproteasomes

The development of immunoproteasome-selective inhibitors is a promising strategy for treating hematologic malignancies, autoimmune and inflammatory diseases. In this context, we report the design, synthesis, and biological evaluation of a new series of amide derivatives as immunoproteasome inhibitors. Notably, the designed compounds act as noncovalent inhibitors, which might be a promising therapeutic option because of the lack of drawbacks and side effects associated with irreversible inhibition. Among the synthesized compounds, we identified a panel of active inhibitors with K_i values in the low micromolar or sub-micromolar ranges toward the β5i and/or β1i subunits of immunoproteasomes. One of the active compounds was shown to be the most potent and selective inhibitor with a K_i value of 21 nm against the single β1i subunit. Docking studies allowed us to determine the mode of binding of the molecules in the catalytic site of immunoproteasome subunits.     

Evaluation of 4-(4-Fluorobenzyl)piperazin-1-yl]-Based Compounds as Competitive Tyrosinase Inhibitors Endowed with Antimelanogenic Effects

There is a considerable attention for the development of inhibitors of tyrosinase (TYR) as therapeutic strategy for the treatment of hyperpigmentation disorders in humans. Continuing in our efforts to identify TYR inhibitors, we describe the design, synthesis and pharmacophore exploration of new small molecules structurally characterized by the presence of the 4-fluorobenzylpiperazine moiety as key pharmacophoric feature for the inhibition of TYR from Agaricus bisporus (AbTYR). Our investigations resulted in the discovery of the competitive inhibitor [4-(4-fluorobenzyl)piperazin-1-yl]-(3-chloro-2-nitro-phenyl)methanone 26 (IC₅₀=0.18 μM) that proved to be ∼100-fold more active than reference compound kojic acid (IC₅₀=17.76 μM). Notably, compound 26 exerted antimelanogenic effect on B16F10 cells in absence of cytotoxicity. Docking analysis suggested its binding mode into AbTYR and into modelled human TYR.     

Bisnaphthalimidopropyl Derivatives as Inhibitors of Leishmania SIR2 Related Protein 1

The NAD⁺‐dependent deacetylases, namely sirtuins, are involved in the regulation of a variety of biological processes such as gene silencing, DNA repair, longevity, metabolism, apoptosis, and development. An enzyme from the parasite Leishmania infantum that belongs to this family, LiSIR2RP1, is a NAD⁺‐dependent tubulin deacetylase and an ADP‐ribosyltransferase. This enzyme's involvement in L. infantum virulence and survival underscores its potential as a drug target. Our search for selective inhibitors of LiSIR2RP1 has led, for the first time, to the identification of the antiparasitic and anticancer bisnaphthalimidopropyl (BNIP) alkyl di‐ and triamines (IC₅₀ values in the single‐digit micromolar range for the most potent compounds). Structure–activity studies were conducted with 12 BNIP derivatives that differ in the length of the central alkyl chain, which links the two naphthalimidopropyl moieties. The most active and selective compound is the BNIP diaminononane (BNIPDanon), with IC₅₀ values of 5.7 and 97.4 μM against the parasite and human forms (SIRT1) of the enzyme, respectively. Furthermore, this compound is an NAD⁺‐competitive inhibitor that interacts differently with the parasite and human enzymes, as determined by docking analysis, which might explain its selectivity toward the parasitic enzyme.     

Phytol Derivatives as Drug Resistance Reversal Agents

Phytol was chemically transformed into fifteen semi‐synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with nalidixic acid against E. coli strains CA8000 and DH5α. The pivaloyl ( 4 ), 3,4,5‐trimethoxybenzoyl ( 9 ), 2,3‐dichlorobenzoyl ( 10 ), cinnamoyl ( 11 ), and aldehyde ( 14 ) derivatives of phytol ((2E,7R,11R)‐3,7,11,15‐tetramethyl‐2‐hexadecen‐1‐ol) were evaluated by using another antibiotic, tetracycline, against the MDREC‐KG4 clinical isolate of E. coli. Derivative 4 decreased the maximal inhibitory concentration (MIC) of the antibiotics by 16‐fold, while derivatives 9 , 10 , 11 , and 14 reduced MIC values of the antibiotics up to eightfold against the E. coli strains. Derivatives 4 , 9 , 10 , 11 , and 14 inhibited the ATP‐dependent efflux pump; this was also supported by their in silico binding affinity and down‐regulation of the efflux pump gene yojI, which encodes the multidrug ATP‐binding cassette transporter protein. This study supports the possible use of phytol derivatives in the development of cost‐effective antibacterial combinations.     

Overcoming Multidrug Resistance (MDR): Design,Biological Evaluation and Molecular Modelling Studies of 2,4-Substituted Quinazoline Derivatives

Some 2,4-disubstituted quinazolines were synthesized and studied as multidrug resistance (MDR) reversers. The new derivatives carried the quinazoline-4-amine scaffold found in modulators of the ABC transporters involved in MDR, as the TKIs gefitinib and erlotinib. Their behaviour on the three ABC transporters, P-gp, MRP1 and BCRP, was investigated. Almost all compounds inhibited the P-gp activity in MDCK-MDR1 cells overexpressing P-gp, showing EC₅₀ values in the nanomolar range ( 1 d , 1 e , 2 a , 2 c , 2 e ). Some compounds were active also towards MRP1 and/or BCRP. Docking results obtained by in silico studies on the P-gp crystal structure highlighted common features for the most potent compounds. The P-gp selective compound 1 e was able to increase the doxorubicin uptake in HT29/DX cells and to restore its antineoplastic activity in resistant cancer cells in the same extent of sensitive cells. Compound 2 a displayed a dual inhibitory effect showing good activities towards both P-gp and BCRP.     

Small-Molecule Inhibitors of Shp2 Phosphatase as Potential Chemotherapeutic Agents for Glioblastoma: A Minireview

Glioblastoma multiforme (GBM) is a dreadful cancer characterised by poor prognosis, low survival rate and difficult clinical correlations. Several signalling pathways and molecular mediators are known to precipitate GBM, and small-molecular targets of these mediators have become a favoured thrust area for researchers to develop potent anti-GBM drugs. Shp2, an important phosphatase of the nonreceptor type protein tyrosine phosphatase (PTPN) subfamily is responsible for master regulation of several such signalling pathways in normal and glioma cells. Thus, inhibition of Shp2 is a logical strategy for the design and development of anti-neoplastic drugs against GBM. Though tapping the full potential of Shp2 binding sites has been challenging, nevertheless, many synthetic and natural scaffolds have been documented as possessing potent and selective anti-Shp2 activities in biochemical and cellular assays, through either active-site or allosteric binding. Most of these scaffolds share a few common pharmacophoric features, a thorough study of which is useful in paving the way for the design and development of improved Shp2 inhibitors. This minireview summarizes the current scenario of potent small-molecule Shp2 inhibitors and emphasizes the anti-GBM potential of some important scaffolds that have shown promising GBM-specific activity in in vitro and in vivo models, thus proving their efficacy in GBM therapy. This review could guide researchers to design new and improved anti-Shp2 pharmacophores and develop them as anti-GBM agents by employing GBM-centric drug-discovery protocols.     

Structural Determinants of CX-4945 Derivatives as Protein Kinase CK2 Inhibitors: A Computational Study

Protein kinase CK2, also known as casein kinase-2, is involved in a broad range of physiological events including cell growth, proliferation and suppression of apoptosis which are related to human cancers. A series of compounds were identified as CK2 inhibitors and their inhibitory activities varied depending on their structures. In order to explore the structure-activity correlation of CX-4945 derivatives as inhibitors of CK2, in the present study, a set of ligand- and receptor-based 3D-QSAR models were developed employing Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA). The optimum CoMFA (R(cv) (2) = 0.618, R(pred) (2) = 0.892) and CoMSIA (R(cv) (2) = 0.681, R(pred) (2) = 0.843) models exhibited reasonable statistical characteristics for CX-4945 derivatives. The results indicated that electrostatic effects contributed the most to both CoMFA and CoMSIA models. The combination of docking analysis and molecular dynamics (MD) simulation showed that Leu45, Lys68, Glu81, Val116, Asp175 and Trp176 of CK2 which formed several direct or water-bridged H-bonds with CX-4945 are crucial for CX-4945 derivatives recognition to CK2. These results can offer useful theoretical references for designing more potent CK2 inhibitors.     

A Library of Thiazolidin-4-one Derivatives as Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors: An Attempt To Discover Novel Antidiabetic Agents

Protein tyrosine phosphatase 1B (PTP1B) is an important target for the treatment of diabetes. A series of thiazolidin-4-one derivatives 8 – 22 was designed, synthesized and investigated as PTP1B inhibitors. The new molecules inhibited PTP1B with IC₅₀ values in the micromolar range. 5-(Furan-2-ylmethylene)-2-(4-nitrophenylimino)thiazolidin-4-one ( 17 ) exhibited potency with a competitive type of enzyme inhibition. structure–activity relationship studies revealed various structural facets important for the potency of these analogues. The findings revealed a requirement for a nitro group-including hydrophobic heteroaryl ring for PTP1B inhibition. Molecular docking studies afforded good correlation with experimental results. H-bonding and π–π interactions were responsible for optimal binding and effective stabilization of virtual protein-ligand complexes. Furthermore, in-silico pharmacokinetic properties of test compounds predicted their drug-like characteristics for potential oral use as antidiabetic agents.Additionally, a binding site model demonstrating crucial pharmacophoric characteristics influencing potency and binding affinity of inhibitors has been proposed, which can be employed in the design of future potential PTP1B inhibitors.