Differences In Inhibitor Binding Research Articles

Structural Studies of Inhibitors with Clinically Relevant Influenza Endonuclease Variants.

Vital to the treatment of influenza is the use of antivirals such as Oseltamivir (Tamiflu) and Zanamivir (Relenza); however, antiviral resistance is becoming an increasing problem for these therapeutics. The RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, a critical component of influenza viral replication machinery, is an antiviral target that was recently validated with the approval of Baloxavir Marboxil (BXM). Despite its clinical success, BXM has demonstrated susceptibility to resistance mutations, specifically the I38T, E23K, and A36 V mutants of PAN. To better understand the effects of these mutations on BXM resistance and improve the design of more robust therapeutics, this study examines key differences in protein-inhibitor interactions with two inhibitors and the I38T, E23K, and A36 V mutants. Differences in inhibitor binding were evaluated by measuring changes in binding to PAN using two biophysical methods. The binding mode of two distinct inhibitors was determined crystallographically with both wild-type and mutant forms of PAN. Collectively, these studies give some insight into the mechanism of antiviral resistance of these mutants.

Structural Characterization of Thiadiazolesulfonamide Inhibitors Bound to Neisseria gonorrhoeae α-Carbonic Anhydrase.

Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.

Pharmacological and molecular dynamics analyses of differences in inhibitor binding to human and nematode PDE4: Implications for management of parasitic nematodes

Novel chemical controls are needed that selectively target human, animal, and plant parasitic nematodes with reduced adverse effects on the host or the environment. We hypothesize that the phosphodiesterase (PDE) enzyme family represents a potential target for development of novel nematicides and anthelmintics. To test this, we identified six PDE families present in the nematode phylum that are orthologous to six of the eleven human PDE families. We characterized the binding interactions of family-selective PDE inhibitors with human and C. elegans PDE4 in conjunction with molecular dynamics (MD) simulations to evaluate differences in binding interactions of these inhibitors within the PDE4 catalytic domain. We observed that roflumilast (human PDE4-selective inhibitor) and zardaverine (selective for human PDE3 and PDE4) were 159- and 77-fold less potent, respectively, in inhibiting C. elegans PDE4. The pan-specific PDE inhibitor isobutyl methyl xanthine (IBMX) had similar affinity for nematode and human PDE4. Of 32 residues within 5 Å of the ligand binding site, five revealed significant differences in non-bonded interaction energies (van der Waals and electrostatic interaction energies) that could account for the differential binding affinities of roflumilast and zardaverine. One site (Phe506 in the human PDE4D3 amino acid sequence corresponding to Tyr253 in C. elegans PDE4) is predicted to alter the binding conformation of roflumilast and zardaverine (but not IBMX) into a less energetically favorable state for the nematode enzyme. The pharmacological differences in sensitivity to PDE4 inhibitors in conjunction with differences in the amino acids comprising the inhibitor binding sites of human and C. elegans PDE4 catalytic domains together support the feasibility of designing the next generation of anthelmintics/nematicides that could selectively bind to nematode PDEs.

Investigating mutation-specific biological activities of small molecules using quantitative structure-activity relationship for epidermal growth factor receptor in cancer

Epidermal Growth Factor Receptor (EGFR) is a potential drug target in cancer therapy. Missense mutations play major roles in influencing the protein function, leading to abnormal cell proliferation and tumorigenesis. A number of EGFR inhibitor molecules targeting ATP binding domain were developed for the past two decades. Unfortunately, they become inactive due to resistance caused by new mutations in patients, and previous studies have also reported noticeable differences in inhibitor binding to distinct known driver mutants as well. Hence, there is a high demand for identification of EGFR mutation-specific inhibitors. In our present study, we derived a set of anti-cancer compounds with biological activities against eight typical EGFR known driver mutations and developed quantitative structure-activity relationship (QSAR) models for each separately. The compounds are grouped based on their functional scaffolds, which enhanced the correlation between compound features and respective biological activities. The models for different mutants performed well with a correlation coefficient, (r) in the range of 0.72–0.91 on jack-knife test. Further, we analyzed the selected features in different models and observed that hydrogen bond and aromaticity-related features play important roles in predicting the biological activity of a compound. This analysis is complimented with docking studies, which showed the binding patterns and interactions of ligands with EGFR mutants that could influence their activities.

Enzyme homologues have distinct reaction paths through their transition states.

Recent studies of the bacterial enzymes EcMTAN and VcMTAN showed that they have different binding affinities for the same transition state analogue. This was surprising given the similarity of their active sites. We performed transition path sampling simulations of both enzymes to reveal the atomic details of the catalytic chemical step, which may be the key for explaining the inhibitor affinity differences. Even though all experimental data would suggest the two enzymes are almost identical, subtle dynamic differences manifest in differences of reaction coordinate, transition state structure, and eventually significant differences in inhibitor binding. Unlike EcMTAN, VcMTAN has multiple distinct transition states, which is an indication that multiple sets of coordinated protein motions can reach a transition state. Reaction coordinate information is only accessible from transition path sampling approaches, since all experimental approaches report averages. Detailed knowledge could have a significant impact on pharmaceutical design.

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity.

The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523–530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7–10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278.

Involvement of DPP‐IV catalytic residues in enzyme–saxagliptin complex formation

The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin: IDENTIFICATION OF RESIDUES INVOLVED IN SUBSTRATE HYDROLYSIS AND INHIBITOR BINDING OF NEPRILYSIN 2

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of the in vitro enzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S'(2) subsite of NEP2 where Ser(133) and Leu(739) replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg(131) and Asn(567) in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S'(2) pocket, particularly the presence therein of Leu(739), account for a number of differences in inhibitor binding between NEP and NEP2.

Crystal analysis of aromatic sulfonamide binding to native and (Zn)2 adduct of human carbonic anhydrase I Michigan 1

The crystal structures of sulfanilamide (4-aminobenzene-sulfonamide) complexed to the enzyme variant human carbonic anhydrase I Michigan 1 (CA I M1) and to its (Zn)2 adduct refined at 2.2 and 2.6 Å resolution respectively are described. Comparisons among these structures and the corresponding sulfonamide adduct of human CA I show significant differences in the orientation of the inhibitor molecule and in its interactions with active site residues such as His200, Thr199, Leu198, Gln92, and Arg/His67 which are known to play important roles in substrate or inhibitor recognition and binding. In CA I M1 molecule B a lengthening of the Zn-N1 sulfanilamide bond distance and a corresponding shortening of the distance between the sulfonamido group and Thr199 are observed compared with the values for native CA I. When the second Zn(II) ion is present in the active site, the p-amino group and the aromatic ring of the inhibitor molecule appear to tilt towards Gln92 and Arg67, moving away from residues His200 and Leu198. The structural differences in inhibitor binding between the CA I isozyme and the CA I M1 variant are discussed in terms of the different inhibition constants measured for a variety of aromatic and heterocyclic sulfonamides.

Crystal Structure of Barley 1,3–1,4-β-Glucanase at 2.0-Å Resolution and Comparison with Bacillus1,3–1,4-β-Glucanase

Both plants and bacteria produce enzymes capable of degrading the mixed-linked beta-glucan of the endosperm cell walls of cereal grains. The enzymes share the specificity for beta-1,4 glycosyl bonds of O-3-substituted glucose units in linear polysaccharides and a similar cleavage mechanism but are unrelated in sequence and tertiary structure. The three-dimensional structure of the 1,3-1, 4-beta-glucanase isoenzyme EII from barley was determined from monoclinic crystals at a resolution of 2.0 A. The protein is folded into a betaalpha8 barrel structure as has been shown previously (Varghese, J. N., Garrett, T. P. J., Colman, P. M., Chen, L., Hoj, P. B., and Fincher, G. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2785-2789) by diffraction analysis at lower resolution of tetragonal crystals. It contains one N-glycosylation site which is described in detail with the sugar moieties attached to residue Asn190. The geometry and hydration of the barley 1,3-1,4-beta-glucanase is analyzed; a model beta-glucan fragment is placed into the binding site by molecular dynamics simulation, and the beta-glucan binding grooves of the plant and bacterial enzymes are compared. Their active sites are shown to have a small number of common features in generally dissimilar geometries that serve to explain both the identical substrate specificity and the observed differences in inhibitor binding.

X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproteinase superfamily

Background: Stromelysin belongs to a family of zinc-dependent endopeptidases referred to as matrix metalloproteinases (MMPs, matrixins) because of their capacity for selective degradation of various components of the extracellular matrix. Matrixins play key roles in diseases as diverse as arthritis and cancer and hence are important targets for therapeutic intervention. Results The crystal structure of the stromelysin catalytic domain (SCD) with bound hydroxamate inhibitor, solved by multiple isomorphous replacement, shows a deep S 1′ specificity pocket which explains differences in inhibitor binding between the collagenases and stromelysin. The binding of calcium ions by loops at the two ends of a β-strand which marks the boundary of the active site provides a structural rationale for the importance of these cations for stability and catalytic activity. Major differences between the matrixins are clustered in two regions forming the entrance to the active site and hence may be determinants of substrate selectivity. Conclusion Structural comparisons of SCD with representative members of the metalloproteinase superfamily clearly highlight the conservation of key secondary structural elements, in spite of major variations in the sequences including insertions and deletions of functional domains. However, the three-dimensional structure of SCD, which is generally closely related to the collagenases, shows significant differences not only in the peripheral regions but also in the specificity pockets; these latter differences should facilitate the rational design of specific inhibitors.

Sequence and structure similarities of cathepsin B from the parasite Schistosoma mansoni and human liver

A three-dimensional structure of Schistosoma mansoni cathepsin B was modelled using the coordinates of the crystal structure of the human liver enzyme. Both enzymes appear to share remarkable structural similarity. However, an examination of the models complexed with two synthetic inhibitors revealed differences in inhibitor binding, as confirmed by differences in the 50% inhibitory concentration of the same inhibitor.