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Tyrosinase is a copper-containing enzyme involved in the biosynthesis of melanin pigment. While the excess production of melanin causes hyperpigmentation of human skin, hypopigmentation results in medical conditions like vitiligo. Tyrosinase inhibitors could be used as efficient skin whitening agents and tyrosinase agonists could be used for enhanced melanin synthesis and skin protection from UV exposure. Among a wide range of tyrosinase-regulating compounds, natural and synthetic derivatives of furochromenones, such as 8-methoxypsoralen (8-MOP), are known to both activate and inhibit tyrosinase. We recently reported a synthetic approach to generate a variety of dihydrofuro[3,2-c]chromenones and furo[3,2-c]chromenones in a metal-free condition. In the present study, we investigated these compounds for their potential as antagonists or agonists of tyrosinase. Using fungal tyrosinase-based in vitro biochemical assay, we obtained one compound (3k) which could inhibit tyrosinase activity, and the other compound (4f) that stimulated tyrosinase activity. The kinetic studies revealed that compound 3k caused 'mixed' type tyrosinase inhibition and 4f stimulated the catalytic efficiency. Studying the mechanisms of these compounds may provide a basis for the development of new effective tyrosinase inhibitors or activators.

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BACKGROUND: Staphylococcus aureus is a common pathogenic microorganism in humans and animals. Type II NADH oxidoreductase (NDH-2) is the only NADH:quinone oxidoreductase present in this organism and represents a promising target for the development of anti-staphylococcal drugs. Recently, myricetin, a natural flavonoid from vegetables and fruits, was found to be a potential inhibitor of NDH-2 of S. aureus. The objective of this study was to evaluate the inhibitory properties of myricetin against NDH-2 and its impact on the growth and expression of virulence factors in S. aureus. RESULTS: A screening method was established to identify effective inhibitors of NDH-2, based on heterologously expressed S. aureus NDH-2. Myricetin was found to be an effective inhibitor of NDH-2 with a half maximal inhibitory concentration (IC50) of 2 µM. In silico predictions and enzyme inhibition kinetics further characterized myricetin as a competitive inhibitor of NDH-2 with respect to the substrate menadione (MK). The minimum inhibitory concentrations (MICs) of myricetin against S. aureus strains ranged from 64 to 128 µg/mL. Time-kill assays showed that myricetin was a bactericidal agent against S. aureus. In line with being a competitive inhibitor of the NDH-2 substrate MK, the anti-staphylococcal activity of myricetin was antagonized by MK-4. In addition, myricetin was found to inhibit the gene expression of enterotoxin SeA and reduce the hemolytic activity induced by S. aureus culture on rabbit erythrocytes in a dose-dependent manner. CONCLUSIONS: Myricetin was newly discovered to be a competitive inhibitor of S. aureus NDH-2 in relation to the substrate MK. This discovery offers a fresh perspective on the anti-staphylococcal activity of myricetin.

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Human carbonic anhydrases (hCAs) play a central role in various physiological processes in the human body. HCAs catalyze the reversible hydration of CO2 into HCO3-, and hence maintains the fluid and pH balance. Overexpression of CA II is associated with diseases, such as glaucoma, and epilepsy. Therefore, CAs are important clinical targets and inhibition of different isoforms, especially hCA II is used in treatment of glaucoma, altitude sickness, and epilepsy. Therapeutically used CA inhibitors (CAI) are sulfonamide-based, such as acetazolamide, dichlorphenamide, methazolamide, ethoxzolamide, etc. However, they exhibit several undesirable effects such as numbness, tingling of extremities, malaise, metallic taste, fatigue, renal calculi, and metabolic acidosis. Therefore, there is an urgent need to identify safe and effective inhibitors of the hCAs. In this study, different phenyl boronic acids 1-5 were evaluated against bovine (bCA II) and hCA II. Among all, compound 1 (4-acetylphenyl boronic acid) was found to be active against bCAII and hCA II with IC50 values of 246 ± 0.48 and 281.40 ± 2.8 µM, respectively, while the remaining compounds were found in-active. Compound 1 was identified as competitive inhibitor of hCA II enzyme (Ki = 283.7 ± 0.002 µM). Additionally, compound 1 was found to be non-toxic against BJ Human fibroblast cell line. The X-ray crystal structure for hCA II in-complex with compound 1 was evaluated to a resolution of 2.6 Å. In fact, this the first structural analysis of a phenyl boron-based inhibitor bound to hCA II, allowing an additional structure-activity analysis of the compounds. Compound 1 was found to be directly bound in the active site of hCA II by interacting with His94, His119, and Thr199 residues. In addition, a bond of 3.11 Å between the zinc ion and coordinated boron atom of the boronic acid moiety of compound 1 was also observed, contributing to binding affinity of compound 1 for hCA II. PDB ID: 8IGF.

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Hyaluronidase is a promising target in drug discovery, given its overexpression in a range of physiological and pathological processes, including tumor migration, skin aging, sagging, and wrinkling, as well as inflammation and bacterial infections. In this study, to identify novel hyaluronidase inhibitors, we applied click chemistry for the modular synthesis of 370 triazoles in 96-well plates, starting with biphenyl azide. Utilizing an optimized turbidimetric screening assay in microplates, we identified Fmoc-containing triazoles 5 and 6, as well as quinoline-containing triazoles 15 and 16, as highly effective hyaluronidase inhibitors. Subsequent research indicated that these triazoles potentially interact with a novel binding site of hyaluronidase. Notably, these inhibitors displayed minimal cytotoxicity and showed promising anti-inflammatory effects in LPS-stimulated macrophages. Remarkably, compound 6 significantly reduced NO release by 74 % at a concentration of 20 µM.

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Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

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The inhibition mode of a retro-inverso (RI) inhibitor containing a hydroxyethylamine dipeptide isostere against the human T-cell leukemia virus type-1 (HTLV-1) protease was examined. Enzymatic evaluation of the RI-modified inhibitor containing a D-allo-Ile residue revealed that HTLV-1 was competitively inhibited. IC50 values of the RI-modified inhibitor and pepstatin A, a standard inhibitor of aspartic proteases, were nearly equivalent.

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Neurodegeneration disorders, such as Alzheimer's disease (AD), have garnered significant attention due to their impact on individuals and society as a whole. Understanding the mechanisms behind these disorders and developing effective therapy strategies is of utmost importance. One potential therapeutic target that has emerged is Rho-associated coiled-coil containing protein kinase 2 (ROCK2), as its accumulation and activity have been closely linked to memory loss. In this report, we present the findings of a recent discovery involving a new molecule that has the ability to competitively inhibit ROCK2 activity. This molecule was identified through the utilization of a DNA-encoded library (DEL) screening platform. Following selection against ROCK2, an off-DNA compound was synthesized and examined to ascertain its inhibitory properties, selectivity, mechanism of action, and binding mode analysis. From the screening, compound CH-2 has demonstrated an IC50 value of 28 nM against ROCK2, while exhibiting a 5-fold selectivity over ROCK1. Further analysis through molecular docking has provided insights into the specific binding modes of this compound. Our findings suggest that DEL selection offers a rapid method for identifying new inhibitors. Among these, the CH-2 compound shows promise as a potential ROCK2 inhibitor and warrants further investigation.

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Soluble epoxide hydrolase (sEH) is a therapeutic target for inflammation. In the present study, we isolated one new (1) and four known (2-5) compounds from the ethyl acetate fraction of hemp seed hulls. Their structures were elucidated as lignanamides via nuclear magnetic resonance and mass spectral analyses. All five compounds inhibited sEH activity, with half-maximal inhibitory concentrations of 2.7 ± 0.3 to 18.3 ± 1.0 µM. These lignanamides showed a competitive mechanism of inhibition via binding to sEH, with ki values below 10 µmol. Molecular simulations revealed that compounds 1-5 fit stably into the active site of sEH, and the key amino acid residues participating in their bonds were identified. It was confirmed that the potential inhibitors 4 and 5 continuously maintained a distance of 3.5 Å from one (Tyr383) and four amino (Asp335, Tyr383, Asn472, tyr516) residues, respectively. These findings provide a framework for the development of naturally derived sEH inhibitors.

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The pursuit of anti-inflammatory agents has led to intensive research on the inhibition of soluble epoxide hydrolase (sEH) and cytokine production using medicinal plants. In this study, we evaluated the efficacy of cis-khellactone, a compound isolated for the first time from the roots of Peucedanum japonicum. The compound was found to be a competitive inhibitor of sEH, exhibiting an IC50 value of 3.1 ± 2.5 µM and ki value of 3.5 µM. Molecular docking and dynamics simulations illustrated the binding pose of (-)cis-khellactone within the active site of sEH. The results suggest that binding of the inhibitor to the enzyme is largely dependent on the Trp336-Gln384 loop within the active site. Further, cis-khellactone was found to inhibit pro-inflammatory cytokines, including NO, iNOS, IL-1ß, and IL-4. These findings affirm that cis-khellactone could serve as a natural therapeutic candidate for the treatment of inflammation.

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Background: Diabetes mellitus (DM) is a chronic disorder, considered to be a major global health challenge in the 21st century. α-Glucosidase enzyme is a well-known drug target to treat Type II DM. Methods: A new library of biphenyl-substituted triazines was synthesized and confirmed by various spectroscopic techniques. Results: All compounds showed potent α-glucosidase inhibitory activity, with IC50 values ranging from 35.35 ± 0.34 to 564.41 ± 0.91 µM, as the standard acarbose, IC50 value of 750.7 ± 0.13 µM. Our in silico study has predicted key interactions with the enzyme's active site. Drug-likeness and absorption, distribution, metabolism, excretion and toxicity were also studied. Conclusion: This study has identified a range of potential hits against the α-glucosidase enzyme that may serve as antidiabetic agents after further investigations.

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Staphylococcus aureus is an opportunistic pathogen that causes variety of infections range from mild skin diseases to life-threatening sepsis. It is also notorious for acquiring resistance to numerous antibiotics. Parvulin-type peptidyl-prolyl cis-trans isomerase (PPiase) domain containing PrsA protein is considered as an essential folding factor for secreted proteins of Gram-positive bacteria. Therefore, it is considered as a potential target for anti-staphylococcal drug discovery. Juglone, plant-derived 1,4-naphthoquinone, shows confirmed antitumor and antibacterial activities. Destruction of bacterial biofilm, inhibition of enzyme expression, degradation of nucleic acids, and other pathways are likely the major possible mechanisms for Staphylococcus aureus inactivation by juglone. Selective inhibition of parvulin type PPiase by juglone has been proven biochemically. However, detail structural information of parvulin-juglone interaction and mechanism of enzymatic inhibition till unexplored. Past hypothesis on inactivation of parvulin type PPiase due to covalent attachment of juglone molecules to its cysteine residues is not acceptable for the S. aureus PrsA parvulin domain as that lacks cysteine. Docking studies showed that juglone binds to the active site residues of S. aureus PrsA parvulin domain involved in enzymatic reaction. Active site conserved histidine residue of parvulin may be involved in juglone interaction as it was found to be the common interactive residue in majority of docking complexes. Data shows Juglone possibly inhibits parvulin type PPiase through competitive inhibition mechanism. Subtle differences of juglone interactions with other orthologous parvulin domains will help to develop semisynthetic drug with higher specificity against S. aureus.

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Dengue fever is a neglected vector-borne disease and is more prevalent in Asia. Currently, no specific treatment is available. Given the time and cost of de novo drug discovery and development, an alternative option of drug repurposing is becoming an effective tool. We screened a library of 1127 pharmacologically active, metabolically stable, and structurally diverse small anticancer molecules to identify inhibitors of the dengue virus (DENV) NS2B/NS3 protease. Enzyme kinetics and inhibition data revealed four B-cell lymphoma 2 inhibitors, that is, ABT263, ABT737, AT101, and TW37, as potent inhibitors of DENV NS2B/NS3 protease, with IC50 values of 0.86, 1.15, 0.81, and 0.89 µM, respectively. Mode of inhibition experiments and computational docking analyses indicated that ABT263 and ABT737 are competitive inhibitors, whereas AT101 and TW37 are noncompetitive inhibitors of the protease. With further evaluation, the identified inhibitors of the DENV NS2B/NS3 protease have the potential to be developed into specific anti-dengue therapeutics.

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Introduction: The root of Cratoxylum cochinchinense has been widely used as Chinese folk medicine to cure fevers, burns, and abdominal complications because it contains various bioactive metabolites such as xanthones, triterpenes, and flavonoids. In this study, we estimated bacterial neuraminidase inhibition with a series of xanthones from C. cochinchinense. BNA has connected to various biological functions such as pathogenic bacteria infection inflammatory process after infection and biofilm formation. Methods: The identification of xanthones (1-6) bearing geranyl and prenyl groups was established by spectroscopic data using UV, IR, NMR, and HREIMS. BNA inhibitory modes of isolated xanthones were investigated by Double-reciprocal plots. Moreover, the competitive inhibitor was evaluated the additional kinetic modes determined by kinetic parameters (k 3, k 4, and K i app). The molecular docking (MD) and molecular dynamics simulations (MDS) studies also provided the critical information regarding the role of the geranyl and prenyl groups against BNA inhibition. Results: A series of xanthones (1-6) appended prenyl and geranyl groups on the A-ring were isolated, and compounds 1-3 were shown to be new xanthones. The analogues within this series were highly inhibited with excellent affinity against bacterial neuraminidase (BNA). A subtle change in the prenyl or geranyl motif affected the inhibitory potency and behavior significantly. For example, the inhibitory potency and binding affinity resulting from the geranyl group on C4: xanthone 1 (IC50 = 0.38 µM, KA = 2.4434 × 105 L·mol-1) were 100-fold different from those of xanthone 3 (IC50 = 35.8 µM, KA = 0.0002 × 105 L·mol-1). The most potent compound 1 was identified as a competitive inhibitor which interacted with BNA under reversible slow-binding inhibition: K i app = 0.1440 µM, k 3 = 0.1410 µM-1s-1, and k 4 = 0.0203 min-1. The inhibitory potencies (IC50) were doubly confirmed by the binding affinities (KA). Discussion: This study suggests the potential of xanthones derived from C. cochinchinense as promising candidates for developing novel BNA inhibitors. Further research and exploration of these xanthones may contribute to the development of effective treatments for bacterial infections and inflammatory processes associated with BNA activity.

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Rapidly accelerated fibrosarcoma (ARAF, BRAF, CRAF) kinase is central to the MAPK pathway (RAS-RAF-MEK-ERK). Inactive RAF kinase is believed to be monomeric, autoinhibited, and cytosolic, while activated RAF is recruited to the membrane via RAS-GTP, leading to the relief of autoinhibition, phosphorylation of key regulatory sites, and dimerization of RAF protomers. Although it is well known that active and inactive BRAF have differential phosphorylation sites that play a crucial role in regulating BRAF, key details are still missing. In this study, we report the characterization of a novel phosphorylation site, BRAFS732 (equivalent in CRAFS624), located in proximity to the C-terminus binding motif for the 14-3-3 scaffolding protein. At the C terminus, 14-3-3 binds to BRAFpS729 (CRAFpS621) and enhances RAF dimerization. We conducted mutational analysis of BRAFS732A/E and CRAFS624A/E and revealed that the phosphomimetic S→E mutant decreases 14-3-3 association and RAF dimerization. In normal cell signaling, dimerized RAF phosphorylates MEK1/2, which is observed in the phospho-deficient S→A mutant. Our results suggest that phosphorylation and dephosphorylation of this site fine-tune the association of 14-3-3 and RAF dimerization, ultimately impacting MEK phosphorylation. We further characterized the BRAF homodimer and BRAF:CRAF heterodimer and identified a correlation between phosphorylation of this site with drug sensitivity. Our work reveals a novel negative regulatory role for phosphorylation of BRAFS732 and CRAFS624 in decreasing 14-3-3 association, dimerization, and MEK phosphorylation. These findings provide insight into the regulation of the MAPK pathway and may have implications for cancers driven by mutations in the pathway.

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Introduction: Alzheimer's disease (AD) is the main type of dementia, caused by the accumulation of amyloid plaques, formed by amyloid peptides after being processed from amyloid precursor protein (APP) by γ- and ß-secretases (BACE-1). Although amyloid peptides have been well established for AD, they have been found in other neurodegenerative diseases, such as Parkinson's disease, Lewy body dementia, and amyotrophic lateral sclerosis. Inhibitors of BACE-1 have been searched and developed, but clinical trials failed due to lack of efficacy or toxicity. Nevertheless, it is still considered a good therapeutic target, as it was proven to remove amyloid peptides and improve memory. Methods: In this work, we designed a peptide based on a sequence obtained from the marine fish Merluccius productus and evaluated it by molecular docking to verify its binding to BACE-1, which was tested experimentally by enzymatic kinetics and cell culture assays. The peptide was injected in healthy mice to study its pharmacokinetics and toxicity. Results: We could obtain a new sequence in which the first N-terminal amino acids and the last one bound to the catalytic site of BACE-1 and showed high stability and hydrophobicity. The synthetic peptide showed a competitive inhibition of BACE-1 and Ki = 94 nM, and when injected in differentiated neurons, it could reduce Aß42o production. In plasma, its half-life is ∼1 h, clearance is 0.0015 µg/L/h, and Vss is 0.0015 µg/L/h. The peptide was found in the spleen and liver 30 min after injection and reduced its level after that, when it was quantified in the kidneys, indicating its fast distribution and urinary excretion. Interestingly, the peptide was found in the brain 2 h after its administration. Histological analysis showed no morphological alteration in any organ, as well as the absence of inflammatory cells, indicating a lack of toxicity. Discussion: We obtained a new BACE-1 inhibitor peptide with fast distribution to the tissues, without accumulation in any organ, but found in the brain, with the possibility to reach its molecular target, BACE-1, contributing to the reduction in the amyloid peptide, which causes amyloid-linked neurodegenerative diseases.

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A significant contributing factor in the development of breast cancer is the estrogens. The synthesis of estrogens is primarily facilitated by aromatase (CYP19), a cytochrome P450 enzyme. Notably, aromatase is expressed at a higher level in human breast cancer tissue compared with the normal breast tissue. Therefore, inhibiting aromatase activity is a potential strategy in hormone receptor-positive breast cancer treatment. In this study, Cellulose Nanocrystals (CNCs) were obtained from Chicory plant waste through a sulfuric acid hydrolysis method with the objective of investigating that whether the obtained CNCs could act as an inhibitor of aromatase enzyme, and prevent the conversion of androgens to estrogens. Structural analysis of CNCs was carried out using Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), while morphological results were obtained using AFM, TEM, and FE-SEM. Furthermore, the nano-particles were found to be spherical in shape with a diameter range of 35-37 nm and displayed a reasonable negative surface charge. Stable transfection of MCF-7 cells with CYP19 has demonstrated the ability of CNCs to inhibit aromatase activities and prevent cell growth by interfering with the enzyme activities. Spectroscopic results revealed the binding constant of CYP19-CNCs and (CYP19-Androstenedione)-CNCs complexes to be 2.07 × 103 L/gr and 2.06 × 104 L/gr, respectively. Conductometry and CD data reported different interaction behaviors among CYP19 and CYP19-Androstenedione complexes at the presence of CNCs in the system. Moreover, the addition of CNCs to the solution in a successive manner resulted in the enhancement of the secondary structure of the CYP19-androstenedione complex. Additionally, CNCs showed a marked reduction in the viability of cancer cells compared to normal cells by enhancing the expression of Bax and p53 at protein and mRNA levels, and by decreasing mRNA levels of PI3K, AKT, and mTOP, as well as protein levels of PI3Kg-P110 and P-mTOP, in MCF-7 cells after incubation with CNCs at IC50 concentration. These findings confirm the decrease in proliferation of breast cancer cells associated with induction of apoptosis through down-regulation of the PI3K/AKT/mTOP signaling pathway. According to the provided data, the obtained CNCs are capable of inhibiting aromatase enzyme activity, which has significant implications for the treatment of cancer.Communicated by Ramaswamy H. Sarma.

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AKT1 is a family of serine/threonine kinases that play a key role in regulating cell growth, proliferation, metabolism, and survival. Two significant classes of AKT1 inhibitors (allosteric and ATP-competitive) are used in clinical development, and both of them could be effective in specific conditions. In this study, we investigated the effect of several different inhibitors on two conformations of the AKT1 by computational approach. We studied the effects of four inhibitors, including MK-2206, Miransertib, Herbacetin, and Shogaol, on the inactive conformation of AKT1 protein and the effects of four inhibitors, Capivasertib, AT7867, Quercetin, and Oridonin molecules on the active conformation of AKT1 protein. The results of simulations showed that each inhibitor creates a stable complex with AKT1 protein, although AKT1/Shogaol and AKT1/AT7867 complexes showed less stability than other complexes. Based on RMSF calculations, the fluctuation of residues in the mentioned complexes is higher than in other complexes. As compared to other complexes in either of its two conformations, MK-2206 has a stronger binding free energy affinity in the inactive conformation, -203.446 kJ/mol. MM-PBSA calculations showed that the van der Waals interactions contribute more than the electrostatic interactions to the binding energy of inhibitors to AKT1 protein.

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Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

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The Mycobacterium tuberculosis low-molecular weight protein tyrosine phosphatase (MptpA) is responsible for the inhibition of phagosome-lysosome fusion and is essential for the bacterium pathogenicity. This inhibition implies that M. tuberculosis is not exposed to a strongly acidic environment in vivo, enabling successful propagation in host cells. Remarkably, MptpA has been previously structurally and functionally investigated, with special emphasis devoted to the enzyme properties at pH 8.0. Considering that the virulence of M. tuberculosis is strictly dependent on the avoidance of acidic conditions in vivo, we analysed the pH-dependence of the structural and catalytic properties of MptpA. Here we show that this enzyme undergoes pronounced conformational rearrangements when exposed to acidic pH conditions, inducing a severe decrease of the enzymatic catalytic efficiency at the expense of phosphotyrosine (pTyr). In particular, a mild decrease of pH from 6.5 to 6.0 triggers a significant increase of K0.5 of MptpA for phosphotyrosine, the phosphate group of which we determined to feature a pKa2 equal to 5.7. Surface plasmon resonance experiments confirmed that MptpA binds poorly to pTyr at pH values < 6.5. Notably, the effectiveness of the MptpA competitive inhibitor L335-M34 at pH 6 does largely outperform the inhibition exerted at neutral or alkaline pH values. Overall, our observations indicate a pronounced sensitivity of MptpA to acidic pH conditions, and suggest the search for competitive inhibitors bearing a negatively charged group featuring pKa values lower than that of the substrate phosphate group.

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The epidermal growth factor receptor (EGFR) plays a critical role in the tumorigenesis of various forms of cancer. Targeting the mutant forms of EGFR has been identified as an attractive therapeutic approach and led to the approval of three generations of inhibitors. The quinazoline core has emerged as a favorable scaffold for the development of novel EGFR inhibitors due to increased affinity for the active site of EGFR kinase. Currently, there are five first-generation (gefitinib, erlotinib, lapatinib, vandetanib, and icotinib) and two second-generation (afatinib and dacomitinib) quinazoline-based EGFR inhibitors approved for the treatment of various types of cancers. The aim of this review is to outline the structural modulations favorable for the inhibitory activity toward both common mutant (del19 and L858R) and resistance-conferring mutant (T790M and C797S) EGFR forms, and provide an overview of the newly synthesized quinazoline derivatives as potentially competitive, covalent or allosteric inhibitors of EGFR.