Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.

Two-Track Virtual Screening Approach to Identify the Dual Inhibitors of Wild Type and C481S Mutant of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is responsible for the pathogenesis of various autoimmune diseases and chronic lymphocytic leukemia. However, the discovery of efficient medicines has seen limited success due to the constitutively active mutants that acquired the drug resistance. To disclose the dual inhibitors against the wild-type BTK and the problematic drug-resistant C481S mutant, a large chemical library was virtually screened with extensive molecular docking simulations using two target proteins. As a consequence of imposing the configurational restraint to make a hydrogen bond in the hinge region of BTK as well as modifying the ligand dehydration term in the scoring function, a total of 20 dual inhibitors were discovered with the range of the associated IC50 values from 2.5 to 15 µM. All these dual inhibitors revealed the inhibitory activity against the C481S mutant to a comparable extent to that measured for the wild type. Among the new inhibitors, N-(3,5-dimethoxyphenyl)-6,7-dimethoxyquinazolin-4-amine (1) appeared to be most suitable as a starting point of the lead optimization due to the highest biochemical potency against the C481S mutant as well as the lowest molecular weight. To increase the potential of a drug candidate, 1 was modified into 6,7-dimethoxy-N-(pyridin-3-yl)quinazolin-4-amine (12) via chemical synthesis so as to possess better physicochemical properties without loss of the biochemical potency. 12 is suggested as a new effective molecular core from which numerous druggable dual inhibitors of the wild-type BTK and the C481S mutant would be derivatized.

Structure-Based Virtual Screening and De Novo Design to Identify Submicromolar Inhibitors of G2019S Mutant of Leucine-Rich Repeat Kinase 2.

Missense mutations of leucine-rich repeat kinase 2 (LRRK2), including the G2019S mutant, are responsible for the pathogenesis of Parkinson's disease. In this work, structure-based virtual screening of a large chemical library was carried out to identify a number of novel inhibitors of the G2019S mutant of LRRK2, the biochemical potencies of which ranged from the low micromolar to the submicromolar level. The discovery of these potent inhibitors was made possible due to the modification of the original protein-ligand binding energy function in order to include an accurate ligand dehydration energy term. The results of extensive molecular docking simulations indicated that the newly identified inhibitors were bound to the ATP-binding site of the G2019S mutant of LRRK2 through the multiple hydrogen bonds with backbone amide groups in the hinge region as well as the hydrophobic interactions with the nonpolar residues in the P-loop, hinge region, and interdomain region. Among 18 inhibitors derived from virtual screening, 4-(2-amino-5-phenylpyrimidin-4-yl)benzene-1,3-diol (Inhibitor 2) is most likely to serve as a new molecular scaffold to optimize the biochemical potency, because it revealed submicromolar inhibitory activity in spite of its low molecular weight (279.3 amu). Indeed, a highly potent inhibitor (Inhibitor 2n) of the G2019S mutant was derived via the structure-based de novo design using the structure of Inhibitor 2 as the molecular core. The biochemical potency of Inhibitor 2n surged to the nanomolar level due to the strengthening of hydrophobic interactions in the ATP-binding site, which were presumably caused by the substitutions of small nonpolar moieties. Due to the high biochemical potency against the G2019S mutant of LRRK2 and the putatively good physicochemical properties, Inhibitor 2n is anticipated to serve as a new lead compound for the discovery of antiparkinsonian medicines.

Quantum Artificial Neural Network Approach to Derive a Highly Predictive 3D-QSAR Model for Blood-Brain Barrier Passage.

A successful passage of the blood-brain barrier (BBB) is an essential prerequisite for the drug molecules designed to act on the central nervous system. The logarithm of blood-brain partitioning (LogBB) has served as an effective index of molecular BBB permeability. Using the three-dimensional (3D) distribution of the molecular electrostatic potential (ESP) as the numerical descriptor, a quantitative structure-activity relationship (QSAR) model termed AlphaQ was derived to predict the molecular LogBB values. To obtain the optimal atomic coordinates of the molecules under investigation, the pairwise 3D structural alignments were conducted in such a way to maximize the quantum mechanical cross correlation between the template and a target molecule. This alignment method has the advantage over the conventional atom-by-atom matching protocol in that the structurally diverse molecules can be analyzed as rigorously as the chemical derivatives with the same scaffold. The inaccuracy problem in the 3D structural alignment was alleviated in a large part by categorizing the molecules into the eight subsets according to the molecular weight. By applying the artificial neural network algorithm to associate the fully quantum mechanical ESP descriptors with the extensive experimental LogBB data, a highly predictive 3D-QSAR model was derived for each molecular subset with a squared correlation coefficient larger than 0.8. Due to the simplicity in model building and the high predictability, AlphaQ is anticipated to serve as an effective computational screening tool for molecular BBB permeability.

Kinase and GPCR polypharmacological approach for the identification of efficient anticancer medicines.

Discovery of an anticancer medicine using a single target protein has often been unsuccessful due to the complexity of pathogenic mechanisms as well as the presence of redundant signaling pathways. In this work, we attempted to find promising anticancer drug candidates by simultaneously targeting casein kinase 1 delta (CK1δ) and muscarinic acetylcholine receptor M3 (M3R). Through the structure-based virtual screening and de novo design with the modified potential function for protein-ligand binding, a series of benzo[4,5]imidazo[1,2-a][1,3,5]triazine-2-amine (BITA) derivatives were identified as CK1δ inhibitors and also as M3R antagonists. The biochemical potencies of these bifunctional molecules reached the nanomolar and low-micromolar levels with respect to CK1δ and M3R, respectively. A common interaction feature in the calculated CK1δ-inhibitor and M3R-antagonist complexes is that the BITA moiety is well-stabilized in the orthosteric site of M3R and the hinge region of CK1δ through the establishment of the three hydrogen bonds and the hydrophobic contacts in the vicinity. The computational and experimental results found in this work exemplify the efficiency of kinase and GPCR polypharmacology in developing anticancer medicines.

Rational Computational Design of Fourth-Generation EGFR Inhibitors to Combat Drug-Resistant Non-Small Cell Lung Cancer.

Although the inhibitors of singly mutated epidermal growth factor receptor (EGFR) kinase are effective for the treatment of non-small cell lung cancer (NSCLC), their clinical efficacy has been limited due to the emergence of various double and triple EGFR mutants with drug resistance. It has thus become urgent to identify potent and selective inhibitors of triple mutant EGFRs resistant to first-, second-, and third-generation EGFR inhibitors. Herein, we report the discovery of potent and highly selective inhibitors of EGFR exon 19 p.E746_A750del/EGFR exon 20 p.T790M/EGFR exon 20 p.C797S (d746-750/T790M/C797S) mutant, which were derived via two-track virtual screening and de novo design. This two-track approach was performed so as to maximize and minimize the inhibitory activity against the triple mutant and the wild type, respectively. Extensive chemical modifications of the initial hit compounds led to the identification of several low-nanomolar inhibitors of the d746-750/T790M/C797S mutant. Among them, two compounds exhibited more than 104-fold selectivity in the inhibition of EGFRd746-750/T790M/C797S over the wild type. The formations of a hydrogen bond with the mutated residue Ser797 and the van der Waals contact with the mutated residue Met790 were found to be a common feature in the interactions between EGFRd746-750/T790M/C797S and the fourth-generation inhibitors. Such an exceptionally high selectivity could also be attributed to the formation of the hydrophobic contact with a Gly loop residue or the hydrogen bond with Asp855 in the activation loop. The discovery of the potent and selective EGFRd746-750/T790M/C797S inhibitors were actually made possible by virtue of the modified protein-ligand binding free energy function involving a new hydration free energy term with enhanced accuracy. The fourth-generation EGFR inhibitors found in this work are anticipated to serve as a new starting point for the discovery of anti-NSCLC medicines to overcome the problematic drug resistance.

Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition.

Structure based virtual screening attempts to discover DUSP1 inhibitors have yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its analogues were synthesized to explore structure activity relationship (SAR) of DUSP1 inhibition.

Development and application of a comprehensive machine learning program for predicting molecular biochemical and pharmacological properties.

We establish a comprehensive quantitative structure-activity relationship (QSAR) model termed AlphaQ through the machine learning algorithm to associate the fully quantum mechanical molecular descriptors with various biochemical and pharmacological properties. Preliminarily, a novel method for molecular structural alignments was developed in such a way to maximize the quantum mechanical cross correlations among the molecules. Besides the improvement of structural alignments, three-dimensional (3D) distribution of the molecular electrostatic potential was introduced as the unique numerical descriptor for individual molecules. These dual modifications lead to a substantial accuracy enhancement in multifarious 3D-QSAR prediction models of AlphaQ. Most remarkably, AlphaQ has been proven to be applicable to structurally diverse molecules to the extent that it outperforms the conventional QSAR methods in estimating the inhibitory activity against thrombin, the water-cyclohexane distribution coefficient, the permeability across the membrane of the Caco-2 cell, and the metabolic stability in human liver microsomes. Due to the simplicity in model building and the high predictive capability for varying biochemical and pharmacological properties, AlphaQ is anticipated to serve as a valuable screening tool at both early and late stages of drug discovery.

Systematic Computational Design and Identification of Low Picomolar Inhibitors of Aurora Kinase A.

Aurora kinase A (AKA) has served as an effective molecular target for the development of cancer therapeutics. A series of potent AKA inhibitors with the (4-methoxy-pyrimidin-2-yl)-phenyl-amine (MPPA) scaffold are identified using a systematic computer-aided drug design protocol involving structure-based virtual screening, de novo design, and free energy perturbation (FEP) simulations. To enhance the accuracy of the virtual screening to find a proper molecular core and de novo design to optimize biochemical potency, we preliminarily improved the scoring function by implementing a reliable hydration energy term. The overall design strategy proves successful to the extent that some inhibitors reveal exceptionally high potency at low picomolar levels; this was achieved by substituting phenyl, chlorine, and tetrazole moieties on the MPPA scaffold. The establishment of bidentate hydrogen bonds with backbone groups in the hinge region appears to be necessary for the high biochemical potency, consistent with the literature X-ray crystallographic data. The picomolar inhibitory activity also stems from the simultaneous formation of additional hydrogen bonds with the side chains of the hinge region and P-loop residues. The FEP simulation results show that the inhibitory activity surges to the low picomolar level because the interactions in the ATP-binding site of AKA become strong by structural modifications enough to overbalance the increase in dehydration cost. Because of the exceptionally high biochemical potency, the AKA inhibitors reported in this study are anticipated to serve as a new starting point for the discovery of anticancer medicine.

Identification of N-(5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl)acetamide derivatives as novel protein tyrosine phosphatase epsilon inhibitors exhibiting anti-osteoclastic activity.

Cytosolic protein tyrosine phosphatase epsilon (cyt-PTPε) plays a central role in controlling differentiation and function of osteoclasts, whose overactivation causes osteoporosis. Based on our previous study reporting a number of cyt-PTPε inhibitory chemical compounds, we carried out a further and extended analysis of our compounds to examine their effects on cyt-PTPε-mediated dephosphorylation and on osteoclast organization and differentiation. Among five compounds showing target selectivity to cyt-PTPε over three other phosphatases in vitro, two compounds exhibited an inhibitory effect against the dephosphorylation of cellular Src protein, the cyt-PTPε substrate. Moreover, these two compounds caused destabilization of the podosome structure that is necessary for the bone-resorbing activity of osteoclasts, and also attenuated cellular differentiation of monocytes into osteoclasts, without affecting cell viability. Therefore, these findings not only verified anti-osteoclastic effects of our cyt-PTPε inhibitory compounds, but also showed that cyt-PTPε expressed in osteoclasts could be a putative therapeutic target worth considering.

The anti-ALS drug riluzole attenuates pericyte loss in the diabetic retinopathy of streptozotocin-treated mice.

Loss of pericytes, considered an early hallmark of diabetic retinopathy, is thought to involve abnormal activation of protein kinase C (PKC). We previously showed that the anti-amyotrophic lateral sclerosis (ALS) drug riluzole functions as a PKC inhibitor. Here, we examined the effects of riluzole on pathological changes in diabetic retinopathy. Pathological endpoints examined in vivo included the number of pericytes and integrity of retinal vessels in streptozotocin (STZ)-induced diabetic mice. In addition, PKC activation and the induction of monocyte chemotactic protein (MCP1) were assessed in diabetic mice and in human retinal pericytes exposed to advanced glycation end product (AGE) or modified low-density lipoprotein (mLDL). The diameter of retinal vessels and the number of pericytes were severely reduced, and the levels of MCP1 and PKC were increased in STZ-induced diabetic mice. Administration of riluzole reversed all of these changes. Furthermore, the increased expression of MCP1 in AGE- or mLDL-treated cultured retinal pericytes was inhibited by treatment with riluzole or the PKC inhibitor GF109203X. In silico modeling showed that riluzole fits well within the catalytic pocket of PKC. Taken together, our results demonstrate that riluzole attenuates both MCP1 induction and pericyte loss in diabetic retinopathy, likely through its direct inhibitory effect on PKC.

Two-track virtual screening approach to identify both competitive and allosteric inhibitors of human small C-terminal domain phosphatase 1.

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg2+ ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg2+ ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations.

Discovery of EGF Receptor Inhibitors That Are Selective for the d746-750/T790M/C797S Mutant through Structure-Based de Novo Design.

Next-generation epidermal growth factor receptor (EGFR) inhibitors against the d746-750/T790M/C797S mutation were discovered through two-track virtual screening and de novo design. A number of nanomolar inhibitors were identified using 2-aryl-4-aminoquinazoline as the molecular core and the modified binding energy function involving a proper dehydration term, which provides important structural insight into the key principles for high inhibitory activities against the d746-750/T790M/C797S mutant. Furthermore, some of these EGFR inhibitors showed a greater than 1000-fold selectivity for the d746-750/T790M/C797S mutant over the wild type, as well as nanomolar activity against the mutant.

Identification of novel protein tyrosine phosphatase sigma inhibitors promoting neurite extension.

Protein tyrosine phosphatase sigma (PTPσ) is a potential target for the therapeutic treatment of neurological deficits associated with impaired neuronal recovery, as this protein is the receptor for chondroitin sulfate proteoglycan (CSPG), which is known to inhibit neuronal regeneration. Through a high-throughput screening approach started from 6400 representative compounds in the Korea Chemical Bank chemical library, we identified 11 novel PTPσ inhibitors that can be classified as flavonoid derivatives or analogs, with IC50 values ranging from 0.5 to 17.5µM. Biochemical assays and structure-based active site-docking simulation indicate that our inhibitors are accommodated at the catalytic active site of PTPσ as surrogates for the phosphotyrosine group. Treatments of these compounds on PC-12 neuronal cells led to the recovery of neurite extension attenuated by CSPG treatment, demonstrating their potential as antineurodegenerative agents.

The Composite Structure and Two-Peak Emission Behavior of a Ca1.5Ba0.5Si5O3N6:Eu(2+) Phosphor.

A Ca1.5Ba0.5Si5O3N6:Eu(2+) phosphor with a monoclinic lattice in the Cm space group exhibiting a composite structure consisting of CaSi2O2N2-like and BaSi6N8O-like structures was examined in terms of structure and luminescence. The luminescent properties of the Ca1.5Ba0.5Si5O3N6:Eu(2+) phosphor could be suitable for light-emitting diode applications since it exhibited a promising yellow (or amber) emission peaking at ∼ 570-590 nm at excitations of 450-460 nm. The present investigation was focused on verifying the composite structure by employing quantum mechanical calculations such as the Hartree-Fock ab initio calculation and a density functional theory calculation along with precise structural and compositional analyses. The two-peak emission behavior ascribed to the composite structure was also examined in terms of continuous wave and time-resolved photoluminescence. In addition, the energy transfer between two activator sites ascribed to the composite structure was examined in detail.

Investigations on recyclisation and hydrolysis in avibactam mediated serine ß-lactamase inhibition.

ß-Lactams inhibit penicillin-binding proteins (PBPs) and serine ß-lactamases by acylation of a nucleophilic active site serine. Avibactam is approved for clinical use in combination with ceftazidime, and is a breakthrough non ß-lactam ß-lactamase inhibitor also reacting via serine acylation. Molecular dynamics (MD) and quantum chemical calculations on avibactam-mediated inhibition of a clinically relevant cephalosporinase reveal that recyclisation of the avibactam derived carbamoyl complex is favoured over hydrolysis. In contrast, we show that analogous recyclisation in ß-lactam mediated inhibition is disfavoured. Avibactam recyclisation is promoted by a proton shuttle, a 'structural' water protonating the nucleophilic serine, and stabilization of the negative charge developed on aminocarbonyl oxygen. The results imply the potential of calculations for distinguishing between bifurcating pathways during inhibition and in generating hypotheses for predicting resistance. The inability of ß-lactams to undergo recyclisation may be an Achilles heel, but one that can be addressed by suitably functionalized reversibly binding inhibitors.

Discovery of Low Micromolar Dual Inhibitors for Wild Type and L1196M Mutant of Anaplastic Lymphoma Kinase through Structure-Based Virtual Screening.

Although anaplastic lymphoma kinase (ALK) is involved in a variety of malignant human cancers, the emergence of constitutively active mutants with drug resistance has rendered it difficult to identify the new medicines for ALK-dependent cancers. To find the common inhibitors of the wild type ALK and the most abundant drug-resistant mutant (L1196M), we performed molecular docking-based virtual screening of a large chemical library in parallel for the two target proteins. As a consequence of augmenting the accuracy of the docking simulation by implementing a sophisticated hydration free energy term in the scoring function, 12 common inhibitors are discovered with the inhibitory activities ranging from submicromolar to low micromolar levels. The results of the binding free energy decomposition indicate that the biochemical potency of ALK inhibitors can be optimized by reducing the dehydration cost for binding to the receptor protein as well as by strengthening the interactions with amino acid residues in the ATP-binding site. The newly identified ALK inhibitors are found to have a little higher inhibitory activity for the L1196M mutant than for the wild type due to the strengthening of the hydrogen bond interactions in the ATP-binding site. Of the 12 common inhibitors, 2-(5-methyl-benzooxazol-2-ylamino)-quinazolin-4-ol (3) is anticipated to serve as a new molecular scaffold to optimize the biochemical potency because it exhibits low micromolar inhibitory activity with respect to both the wild type and L1196M mutant in spite of the low molecular weight (292.3 amu).

Extended solvent-contact model approach to blind SAMPL5 prediction challenge for the distribution coefficients of drug-like molecules.

The performance of the extended solvent-contact model has been addressed in the SAMPL5 blind prediction challenge for distribution coefficient (LogD) of drug-like molecules with respect to the cyclohexane/water partitioning system. All the atomic parameters defined for 41 atom types in the solvation free energy function were optimized by operating a standard genetic algorithm with respect to water and cyclohexane solvents. In the parameterizations for cyclohexane, the experimental solvation free energy (ΔG sol ) data of 15 molecules for 1-octanol were combined with those of 77 molecules for cyclohexane to construct a training set because ΔG sol values of the former were unavailable for cyclohexane in publicly accessible databases. Using this hybrid training set, we established the LogD prediction model with the correlation coefficient (R), average error (AE), and root mean square error (RMSE) of 0.55, 1.53, and 3.03, respectively, for the comparison of experimental and computational results for 53 SAMPL5 molecules. The modest accuracy in LogD prediction could be attributed to the incomplete optimization of atomic solvation parameters for cyclohexane. With respect to 31 SAMPL5 molecules containing the atom types for which experimental reference data for ΔG sol were available for both water and cyclohexane, the accuracy in LogD prediction increased remarkably with the R, AE, and RMSE values of 0.82, 0.89, and 1.60, respectively. This significant enhancement in performance stemmed from the better optimization of atomic solvation parameters by limiting the element of training set to the molecules with experimental ΔG sol data for cyclohexane. Due to the simplicity in model building and to low computational cost for parameterizations, the extended solvent-contact model is anticipated to serve as a valuable computational tool for LogD prediction upon the enrichment of experimental ΔG sol data for organic solvents.

Discovery of Dual Inhibitors for Wild Type and D816V Mutant of c-KIT Kinase through Virtual and Biochemical Screening of Natural Products.

Although stem cell factor receptor (c-KIT) kinase is responsible for various malignant human cancers, the presence of constitutively active gain-of-function mutants has made it difficult to discover new anticancer agents using c-KIT as the target protein. To identify the common inhibitors of wild-type c-KIT and the most abundant gain-of-function mutant (D816V), the virtual screening of natural products was performed for the two target proteins in parallel with the scoring function improved by implementing a sophisticated solvation free energy term. As a result, four common inhibitors of natural origin are found with biochemical potencies ranging from low micromolar to submicromolar levels. The results of extensive docking simulations show that although the natural-product inhibitors establish weaker hydrophobic interactions with the D816V mutant than with the wild type, they exhibit a little higher inhibitory activity for the former than the latter by strengthening the hydrogen-bond interactions to a sufficient extent. Of the four natural-product inhibitors, (Z)-6-hydroxy-2-(4-methoxybenzylidene)benzofuran-3(2H)-one (3) is anticipated to serve as a new molecular core for the structure-activity relationship studies to optimize the biochemical potencies because it exhibits good inhibitory activity against both the wild type and D816V mutant despite its low molecular weight (268.3 amu).