On the recognition of Yersinia protein tyrosine phosphatase by carboxylic acid derivatives.

Some members of Yersinia (Y), a genus of bacteria in the family Yersiniaceae, are pathogenic in humans, causing a range of health problems, from gastrointestinal syndromes to the plague. The Y protein tyrosine phosphatase (PTP) YopH is a crucial virulence determinant, considering the vital roles of PTPs in the intracellular signal transduction pathways and cell cycle control. The structural understanding of YopH as a cellular target in pathogenic conditions caused by Y infection is a prerequisite for designing potent and selective YopH inhibitors. Thus, by using molecular docking simulations, the open and closed conformations of the so-called 'WPD loop' (352-Gly-Asn-Trp-Pro-Asp-Gln-Thr-Ala-Val-Ser-361), located nearby the active site (403-Cys-Arg-Ala-Gly-Val-Gly-Arg-Thr-410) in YopH structure, are shown to be relevant for recognition by carboxylic acid derivatives, and the closed conformation is a more preferable receptor in terms of the quantitative correlation with experimental data. In both cases, aurintricarboxylic acid (ATA) has the greatest affinity to YopH. Consequently, a quantum mechanics/molecular mechanics (QM/MM) molecular model is derived to see into the extent of the ATA-induced open-closed conformational change. Active site residues and the WPD loop, as well as ATA are treated using SCC-DFTB-D (QM level), while the rest of the complex is treated using AMBER force field (MM level). The active/inactive functional behavior of YopH is explored by observing the interaction mode of ATA with the wild-type (wt)/Cys403Ser receptor and evaluating the competitive inhibition parameters. Implications of the present study for experimental research are discussed. Communicated by Ramaswamy H. Sarma.

On the inhibition of cytochrome P450 3A4 by structurally diversified flavonoids.

Cytochrome P450 3A4 (CYP3A4) is the most versatile enzyme involved in drug metabolism. The time-dependent inhibition of CYP3A4 by acacetin, apigenin, chrysin, and pinocembrin was experimentally detected, but not entirely elaborated so far. Thus, a two-level QM/MM (Quantum Mechanics/Molecular Mechanics) model is developed to yield insights into the receptor-flavonoid recognition at the molecular scale. Active site residues and the flavonoid are modelled using SCC-DFTB-D (QM level), while the rest of the complex is treated using AMBER force field (MM level). QM/MM binding free energies are well correlated with experimental data, indicating the largest inhibitory effect of chrysin on enzyme activity at a submicromolar concentration. Consequently, quercetin (QUE) and flavopiridol (FLP) are observed as representative examples of structurally different flavonoids. The inhibition parameters for QUE and FLP are evaluated using the well-calibrated QM/MM strategy, thereby aiding to quantitatively conceive the functional behavior of the whole family of flavonoids. A kinetic threshold for further assessment of the drug-drug interactions underlying the time-dependent inhibition of CYP3A4 by flavonoids is explored.Communicated by Ramaswamy H. Sarma.

Prediction of Hit-to-Lead Ligand Molecule Interaction with G-Quadruplex DNA from c-Myc Oncogene Promoter Region.

Targeting guanine (G)-rich DNA sequences, folded into non-canonical G-quadruplex (G4) structures, by small ligand molecules is a potential strategy for gene therapy of cancer disease. BRACO-19 has been recently established as a unique (thermodynamically favorable and highly selective) binder, being involved in the external stacking mode of interaction with a G4-DNA formed in the c-Myc oncogene promoter region (P. M. Mitrasinovic, Croat. Chem. Acta 2019, 92, 43-57). Herein, hit-to-lead ligands are identified using high-throughput virtual screening (HTVS). Search of the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases is performed using the key pharmacophore features of BRACO-19. At the very outset, out of a total of 29,009 entries, 95 hits are extracted and evaluated by docking them in the binding sites of G4. Then, 22 hits are chosen by observing the binding free energies. Consequently, 3 hit-to-lead candidates are selected on the basis of structural criteria. Finally, a lead candidate structure is proposed using analog design and considering both the physicochemical requirements for optimal biological activity and a variety of pharmacological standpoints. Implications of the present study for experimental research are discussed.

G-Quadruplexes: Emerging Targets for the Structure-Based Design of Potential Anti-Cancer and Antiviral Therapies.

G-quadruplexes (G4s) are noncanonical secondary structures that fold within guanine (G) rich strands of regulatory genomic regions. Recent evidences suggest their intimate involvement in important biological processes such as telomere maintenance, end-capping and protection, chromosome stability, gene expression, viral integration, and recombination. Mechanistic details of how and why G4 structures influence biological function indicate a rationale for treating G4s as emerging molecular targets for future therapeutics. In other words, the structural heterogeneity with well-defined binding sites, thermal stability and abundance of G4s in telomeres, oncogene promoter regions, and viral genomes make G4s attractive targets for small molecules, aimed to selectively recognize them over all other nucleic acids structures, particularly duplex forms that are most abundant in the genome. Herein, a critical survey of well-characterized G4-interactive ligands as potential tools in anti-cancer and antiviral therapies is presented. Effects that these ligands selectively exert in vitro and in vivo models are summarized. Unique ligands involved in specific G4 recognition are put forward. A key question, how to design and develop new G4 specific ligands that conform to the structural and physicochemical requirements for optimal biological activity, is discussed by considering both remarkable advances over the last few years and our recent contributions.

Prediction of Single Point Mutations in Human Coronavirus and Their Effects on Binding to 9-O-Acetylated Sialic Acid and Hidroxychloroquine.

Due to the current spreading of the new disease CoViD-19, the World Health Organization formally declared a world pandemic on March 11, 2020. The present trends indicate that the pandemic will have an enormous clinical and economic impact on population health. Infections are initiated by the transmembrane spike (S) glycoproteins of human coronavirus (hCoV) binding to host receptors. Ongoing research and therapeutic product development are of vital importance for the successful treatment of CoViD-19. To contribute somewhat to the overall effort, herein, single point mutations (SPMs) of the binding site residues in hCoV-OC43 S that recognizes cellular surface components containing 9-O-acetylated sialic acid (9-O-Ac-Sia) are explored using an in silico protein engineering approach, while their effects on the binding of 9-O-Ac-Sia and Hidroxychloroquine (Hcq) are evaluated using molecular docking simulations. Thr31Met and Val84Arg are predicted to be the critical - most likely SPMs in hCoV-OC43 S for the binding of 9-O-Ac-Sia and Hcq, respectively, even though Thr31Met is a very likely SPM in the case of Hcq too. The corresponding modes of interaction indicate a comparable strength of the Thr31Met/9-O-Ac-Sia and Val84Arg/Hcq (or Thr31Met/Hcq) complexes. Given that the binding site is conserved in all CoV S glycoproteins that associate with 9-O-acetyl-sialoglycans, the high hydrophobic affinity of Hcq to hCoV-OC43 S speaks in favor of its ability to competitively inhibit rapid S-mediated virion attachment in high-density receptor environments, but its considerably low specificity to hCoV-OC43 S may be one of the key obstacles in considering the potential of Hcq to become a drug candidate.

Quantum Mechanics/Molecular Mechanics Study on Caspase-2 Recognition by Peptide Inhibitors.

For a variety of biological and medical reasons, the ongoing development of humane caspase-2 inhibitors is of vital importance. Herein, a hybrid (Quantum Mechanics/Molecular Mechanics - QM/MM), two-layered molecular model is derived in order to understand better the affinity and specificity of peptide inhibitor interaction with caspase-2. By taking care of both the unique structural features and the catalytic activity of human caspase-2, the critical enzyme residues (E217, R378, N379, T380, and Y420) with the peptide inhibitor are treated at QM level (the Self-Consistent-Charge Density-Functional Tight-Binding method with the Dispersion correction (SCC-DFTB-D)), while the remaining part of the complex is treated at MM level (AMBER force field). The QM/MM binding free energies (BFEs) are well-correlated with the experimental observations and indicate that caspase-2 uniquely prefers a penta-peptide such as VDVAD. The sequence of VDVAD is varied in a systematic fashion by considering the physicochemical properties of every constitutive amino acid and its substituent, and the corresponding BFE with the inhibition constant (Ki) is evaluated. The values of Ki for several caspase-2:peptide complexes are found to be within the experimental range (between 0.01 nM and 1 ?M). The affinity order is: VELAD (Ki=0.081 nM) > VDVAD (Ki=0.23 nM) > VEIAD (Ki=0.61 nM) > VEVAD (Ki=3.7 nM) > VDIAD (Ki=4.5 nM) etc. An approximate condition needed to be satisfied by the kinetic parameters (the Michaelis constant - KM and the specificity constant - kcat/KM) for competitive inhibition is reported. The estimated values of kcat/KM, being within the experimentally established range (between 10-4 and 10-1 ?M-1 s-1), indicate that VELAD and VDVAD are most specific to caspase-2. These two particular peptides are nearly 1.5, 3 and 4 times more specific to the receptor than VEIAD, VEVAD and VDIAD respectively. Additional kinetic threshold, aimed to discriminate tightly bound inhibitors, is given.

Structural insights into the binding of small ligand molecules to a G-quadruplex DNA located in the HIV-1 promoter.

Targeting guanine (G)-rich DNA sequences, folded into non-canonical G-quadruplex (G4) structures, by small ligand molecules is a promising strategy for gene therapy of various diseases. There is experimental proposal that, among eight studied ligands, nitidine chloride - NC and a benzo phenanthridine derivative - BPD have the highest binding affinity for such a sequence (5'-T1G2G3C4C5T6G7G8G9C10G11G12G13A14C15T16G17G18G19-3') in the HIV-1 promoter, indicating that an anti-HIV-1 prodrug may regulate the expression of the promoter. Herein, this experimental indication is elaborated by using molecular docking simulations and by characterizing the modes of binding of the eight natural molecules to the particular G4. Moreover, the configurational entropy, as an upper bound of the true entropy contribution to the free energy in noncovalent binding, is employed to see into the structural changes experienced by the G4-DNA upon ligand binding. For various parts (complete structure, backbone, system of all bases, bases of G-tetrads) of the G4-DNA structure, a subtle molecular dynamics (MD) is exploited to determine the change of asymptotic (for infinitely long MD simulation) configurational entropy, being the thermodynamic consequence of DNA flexibility change upon complex formation. While NC increases rigidity of G4 (mainly through the system of all nucleobases), BPD increases flexibility of G4 (more than 50% stems from the sugar-phosphate backbone). These insights are further dissected and substantiated by considering the configurational entropy contributions at the level of individual base pairs making the two G-tetrads (G2G7G13G17 and G3G8G12G18) and by exploring the estimates of the total intra-base pair and inter-base pair entropies. This work makes the structural origin of enhanced stability of G4-DNA more certain - useful information when attempting to design optimal G4-DNA binders.

Sequence-dependent binding of flavonoids to duplex DNA.

The whole family of structurally distinct flavonoids has been recognized as a valuable source of prospective anticancer agents. There is experimental evidence demonstrating that some flavonoids, like flavopiridol (FLP) and quercetin (QUE), bind to DNA influencing their key physiological function. FLP is involved in the combined mode of interaction (intercalation and minor groove binding), while QUE is viewed as a minor groove binder. From a physical standpoint, experimental and theoretical studies have not so far provided a sufficiently consistent picture of the nature of interaction with DNA. Herein the sequence-dependent binding of FLP and of QUE (two representative examples of the structurally different flavonoids) with duplex DNA, containing a variety of the sequences of eight nucleotides (I: GGGGCCCC, II: GGCCGGCC, III: AAAATTTT, IV: AAGCGCTT, V: GCGCGCGC) in the 5'-strand, is investigated using a sophisticated molecular dynamics (MD) approach. For various parts (helix, backbone, bases) of the DNA structure, the change of asymptotic (in terms of an infinite length of MD simulation) configurational entropy, being the thermodynamic consequence of DNA flexibility change due to ligand binding, is explored. As far as the sequence-dependent extent of DNA flexibility change upon QUE (or FLP) binding is concerned, for the entire double helix, increased flexibility is observed for I (or I ≈ II), while increased rigidity is found to be in the order of V > III > II > IV (or III > V > IV) for the rest of sequences. For the backbone, increased rigidity in the order of V > III > II > IV > I (or III > V > IV > I > II) is generally observed. For the nucleobases, increased flexibility is determined for I and II (I > II for both ligands), while increased rigidity in the order of V ≈ III > IV (or III > V > IV) is reported for the other sequences. Of the overall increased rigidity of the DNA structure upon ligand binding that is observed for the sequences III, IV, and V, about 50-70% comes from the sugar-phosphate backbone. Noteworthy is that the increased flexibility of the entire double helix and of the complete system of nucleobases upon ligand binding is only established for sequence I. The insights are further subtly substantiated by considering the configurational entropy contributions at the level of individual nucleobase pairs and of individual nucleo-base pair steps and by analyzing the sequence dependent estimates of intra-base pair entropy and inter-base pair entropy. The GGC triplet, which is part of the central tetramer (GGCC) of I, is concluded to be critical for binding of flavonoids, while the effect of the presence of ligand to the flexibility of nucleobases is localized through the intra-base pair motion of the intercalation site and its immediate vicinity. G-rich DNA sequences with consecutive Gs going before and/or after the critical GGC code (such as I: GGGGCCCC) are proposed to be uniquely specific for flavonoids. The configurational entropy contribution, as an upper bound of the true entropy contribution to the free energy in noncovalent binding, is demonstrated to influence the fundamental discrimination (intercalation vs groove binding) of DNA-flavonoid recognition modes. Some interesting implications for the structure-based design of optimal DNA binders are discussed.

Inhibitory activity against epidermal growth factor receptor (EGFR) based on single point mutations of active site residues.

Epidermal growth factor receptors belong to the ErbB family of receptor tyrosine kinases (TKs) involved in the proliferation of normal and malignant cells. EGFR has attracted considerable attention as a target for cancer therapy. The findings reported herein are believed to provide some novel insights into the design of effective drugs for the therapeutic treatment of EGFR-related cancers. In particular, it is shown using sophisticated computational tools in a systematic way that the affinity of a wide spectrum of thiazolo[4,5-d]pyrimidine analogs can be carefully tuned up by seeking the desired goal in the structural modifications of EGFR, such as single point mutations of the critical EGFR residues in the active site. It is also demonstrated that a large number of the small ligand molecules can be efficiently divided into subgroups of the structurally similar ligands and that every such a subgroup has its unique inhibitory activity signature. The protein engineering approach, as quite reproducible, is proposed to be a viable partner to experiment in addressing a variety of issues, including investigation of clinically important mutations, development of drug resistance, identification of the most promising anti-cancer drug candidates, etc.

Structural elucidation of unique inhibitory activities of two thiazolo[ 4,5-d]pyrimidines against epidermal growth factor receptor (EGFR): implications for successful drug design.

In our previous study, a protein engineering approach, accounting for the effects of single point mutations of the binding site residues on the stability of 22 thiazolo[4,5-d]pyrimidines in complex with the intracellular kinase domain of EGFR (PDB ID: 1XKK), was established in a systematic manner to be an efficient strategy for the identification of anti-EGFR-related-cancer drug candidates. The inhibitory activities of two lignad molecules, 4-(7-(3-chloro-4-morpholinophenylamino)thiazolo[4,5-d]pyrimidin-2-ylamino)benzenesulfonamide and 4-(7-(4-morpholinophenylamino) thiazolo[4,5-d]pyrimidin-2-ylamino)benzenesulfonamide, exhibited some sort of uniqueness. Regardless of a slight mutual structural difference between these two ligands in only a peripheral Cl atom, their inhibitory activities against EGFR appeared to be associated with two quite opposite structural bases respectively. Herein, the fundamental rationalization of the remarkable standpoint is elaborated using both molecular docking and molecular dynamics simulations. Consequently, a number of implications of vital importance for the successful structure-based design of prospective drugs against EGFR-related cancers are discussed.

Progress in structure-based design of EGFR inhibitors.

Epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs) involved in the proliferation of normal and malignant cells. As mutations and overexpression of ErbB TKs are implicated in carcinoma and glioblastoma and are related to both a very strong resistance to chemotherapy and a poor survival means that ErbB receptors are targets of considerable importance for anti-cancer drug design. Besides using monoclonal antibodies for anti-EGFR-related cancer therapeutics, small molecules - tyrosine kinase inhibitors are being considered as well. Some of these therapies have entered clinical trials or have been approved for clinical use. Based on experimental methods (radiometry, immunofluoroscence or luminescence, electrophoresis) that are mainly employed for measuring and interpreting the selectivity of protein kinase inhibitors, routine accomplishment of selectivity of small molecules for particular protein kinases is a substantial challenge. In light of this, we herein elaborate a computer-based protein engineering approach demonstrating its potential to be a viable supplement to experiment in modulating the affinity of ligand molecules for EGFR in an efficient manner. The structural basis of the remarkable strategy is also elucidated using our recent results obtained by means of molecular docking and molecular dynamics simulations. A few critical implications for successful structure-based design of prospective drug candidates against EGFR-related cancers are consequently discussed.

Some implications of receptor kinase signaling pathway for development of multitargeted kinase inhibitors.

Epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs). Based on the role of EGFR signaling pathway in malignant progression of various types of tumors, a growing interest in the use of EGFR-TK inhibitors as probes for molecular imaging of EGFR-overexpressing tumors via positron emission tomography (PET) and single photon emission computed tomography (SPECT) is being notable. On one side, such noninvasive and repetitive monitoring of the activity of EGFR at the kinase level is intended to provide a direct measure of EGFR occupancy and inhibition by EGFR-targeting drugs. On the other side, all oncologic imaging tracers are molecularly targeted radiopharmaceuticals, which are strongly dependent on the tumor biochemistry including increased metabolism, hyperproliferation, angiogenesis, hypoxia, apoptosis, and specific tumor biomarkers (tumor specific antigens and tumor-specific receptors). The present article is an attempt to reconcile these two vital standpoints influencing the choice of appropriate radiolabeled agents for PET and SPECT imaging aimed to support the development of a new generation of multi-targeted kinase inhibitors in the time ahead, because the routine accomplishment of drug selectivity for particular protein kinases is a substantial challenge.

Experimental and theoretical advances in functional understanding of flavonoids as anti-tumor agents.

The potential of flavonoids to act as anti-tumor agents has been recognized but not fully understood because flavonoids are acting at several stages in cancer progression with distinct structure-function relationships. A whole family of structurally different flavonoids is herein described by reviewing some critical aspects of their pro-oxidant behavior in vitro/vivo and in cell systems by which they may work as antioxidants. Different classes of flavonoids (chalcones, flavones, isoflavones, flavanols, flavanones and anthocyanins) are synthetically mimicked using natural product structure-antioxidant activity relationships that are relevant for their enhanced function against cancer as well as severe inflammation conditions under which an increased oxidative stress is often implicated. In the context of the common mechanisms of flavonoid action, clinical data on benefits of flavonoids in fighting against cancer are discussed. A structural basis needed to improve antioxidant activity of these agents is elaborated in more detail.

On the affinity and specificity of quercetin for DNA.

The potential of quercetin (QUE), being a member of the whole family of structurally different flavonoids, to serve as an anti-tumor agent has been recognized, but not fully understood. The interactions between DNA and a series of the flavonoids have so far been mainly investigated using a variety of experimental techniques. Herein, the specificity of QUE for DNA is explored using sophisticated density functional theory (DFT) methods employed to generate the optimized structure of QUE in complex with adenine (A), guanine (G), thymine (T) and cytosine (C), respectively. As far as a preference of QUE is concerned, structural and energetic as well as NMR chemical shift arguments clearly indicate a highest for G and a lowest for C. This observation is further substantiated by analyzing the binding modes of QUE docked in a quadruplex receptor structure of DNA and in a duplex receptor structure of DNA. Among all possible single point mutations of the DNA quadruplex and duplex residues, several critical ones causing a conspicuous stabilizing effect on the original complexes of QUE with the DNA receptors are identified. Consequently, several fundamental standpoints shedding new light on the molecular mechanisms underlying the interactions between QUE and DNA are discussed.

Epidermal growth factor receptors: a functional perspective.

The epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs) involved in the proliferation of normal and malignant cells. EGFR has attracted considerable attention as a target for cancer therapy. This article considers various functional roles of EGFR-based systems that are relevant for the early detection and staging of cancers overexpressing EGFR.

Modeling of HIV-1 TAR RNA-ligand complexes.

Trans activation response (TAR) region is an RNA target of considerable importance in controlling the replication cycle of the human immunodeficiency virus (HIV). At a transcriptional level, HIV-1 is regulated by means of the interaction between Tat protein and TAR RNA. The TAR-Tat complex is an attractive target for developing novel antiviral drugs. Herein, the recognition modes of 8 structurally different ligands, as mimics of Tat protein, in complex with a TAR RNA are investigated using the DOCK 6.4 flexible docking protocol in association with the newly-implemented scoring function AMBER including solvation implicitly through the generalized Born solvent-accessible surface area (GB/SA) continuum model. The TAR RNA-ligand interactions are further characterized and contrasted using the nature of separate contributions to the stability of the complexes. Several interesting implications for the key challenge, the development of low molecular weight ligands binding to HIV-1 TAR RNA with high affinity and specificity, are discussed.

Advances in the structure-based design of the influenza A neuraminidase inhibitors.

Since 2003, highly pathogenic H5N1 influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. At present, there are only three licensed anti-Influenza drugs namely Relenza (Zanamivir - ZMV), Tamiflu (Oseltamivir - OTV) and Amantadine/Rimantadine. The latter targets the M2 ion channel whereas the other compounds target neuraminidase (NA) and were designed through structure-based enzyme inhibitor programmes. Some structural knowledge of the Influenza neuraminidase is now known, due to remarkable advances in crystallographic techniques. The structure of H5N1 NA is particularly attractive because it offers new opportunities for drug design. Besides a profound impact that structural biology has had on understanding the Influenza virus and the rational design of antivirals, computational methods are now a viable partner to experiment in designing NA inhibitors. We herein discuss the development of current neuraminidase inhibitors, the emergence of resistance to them, and recent research progress towards the development of new inhibitors.

On the structure-based design of novel inhibitors of H5N1 influenza A virus neuraminidase (NA).

The structure-based design of novel H5N1 neuraminidase inhibitors is currently a research topic of vital importance owing to both a recent pandemic threat by the worldwide spread of H5N1 avian influenza and the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir-OTV (Tamiflu). A specific criterion used in this work for determining fully acceptable conformations of potential inhibitors is a previous experimental proposal of exploiting potential benefits for drug design offered by the '150-cavity' adjacent to the NA active site. Using the crystal structure of H5N1 NA (PDB ID: 2hty) as the starting point, in a set of 54 inhibitors previously proposed by modifying the side chains of oseltamivir, 4 inhibitors were identified using two different computational strategies (ArgusLab4.0.1, FlexX-E3.0.1) both to lower the binding free energy (BFE) of oseltamivir and to have partially acceptable conformations. These 4 oseltamivr structure-based analogues were found to adopt the most promising conformations by identifying the guanidinium side chain of Arg156 as a prospective partner for making polar contacts, but none of the modified 4-amino groups of oseltamivir in the 4 favorable conformations was found to make polar contacts with the guanidinium side chain of Arg156. Hence, the structures of two additional inhibitors were designed and shown to further lower the binding free energy of OTV relative to the previous 54 inhibitors. These two novel structures clearly suggest that it may be possible for a new substituent to be developed by functional modifications at position of the 4-amino group of oseltamivir in order to make polar contacts with the guanidinium side chain of Arg156, and thereby enhance the binding of a more potent inhibitor. Several standpoints of vital importance for designing novel structures of potentially more effective H5N1 NA inhibitors are established.

Another look at the molecular mechanism of the resistance of H5N1 influenza A virus neuraminidase (NA) to oseltamivir (OTV).

In the context of a recent pandemic threat by the worldwide spread of H5N1 avian influenza, the high resistance of H5N1 virus to the most widely used commercial drug, oseltamivir (Tamiflu), is currently an important research topic. Herein, molecular bases of the mechanism of H5N1 NA resistance to oseltamivir were elucidated using a computational approach in a systematic fashion. Using the crystal structure of the complex of H5N1 NA with OTV (PDB ID: 2hu0) as the starting point, the question, how mutations at His274 by both smaller side chain (Gly, Ser, Asn, Gln) and larger side chain (Phe, Tyr) residues influence the sensitivity of N1 to oseltamivir, was addressed and correlated with the experimental data. The smaller side chain residue mutations of His274 resulted in slightly enhanced or unchanged NA sensitivity to OTV, while His274Phe and His274Tyr reduced the susceptibility of OTV to N1. In contrast to the binding free energies, the net charges of Glu276 and Arg224, making charge-charge interactions with Glu276, were established to be more sensitive to detecting subtle conformational differences induced at the key residue Glu276 by the His274X mutations. This study provides deeper insights into the possibility of developing viable drug-resistant mutants.