Structure-based drug design: exploring the proper filling of apolar pockets at enzyme active sites

The proper filling of apolar pockets at enzyme active sites is central for increasing binding activity and selectivity of hits and leads in medicinal chemistry. In our structure-based design approach toward the generation of potent enzyme inhibitors, we encountered a variety of challenges in gaining suitable binding affinity from the occupation of such pockets. We summarize them here for the first time. A fluorine scan of tricyclic thrombin inhibitors led to the discovery of favorable orthogonal dipolar C-F...CO interactions. Efficient cation-pi interactions were established in the S4 pocket of factor Xa, another serine protease from the blood coagulation cascade. Changing from mono- to bisubstrate inhibitors of catechol O-methyltransferase, a target in the L-Dopa-based treatment of Parkinson's disease, enabled the full exploitation of a previously unexplored hydrophobic pocket. Conformational preorganization of a pocket at an enzyme active site is crucial for harvesting binding affinity. This is demonstrated for two enzymes from the nonmevalonate pathway of isoprenoid biosynthesis, IspE and IspF, which are pursued as antimalarial targets. Disrupting crystallographically defined water networks on the way into a pocket might cost all of the binding free enthalpy gained from its occupation, as revealed in studies with tRNA-guanine transglycosylase, a target against shigellosis. Investigations of the active site of plasmepsin II, another antimalarial target, showed that principles for proper apolar cavity filling, originally developed for synthetic host-guest systems, are also applicable to enzyme environments.     

Molecular insight into the interaction mechanisms of amino‐2H‐imidazole derivatives with BACE1 protease: A QM/MM and QTAIM study

In this study, we described quantitatively the interactions between two new amino‐2H‐imidazole inhibitors ((R)‐1t and (S)‐1m) and BACE1 using a hybrid quantum mechanics‐molecular mechanical (QM/MM) method together with a quantum theory of atoms In Molecules (QTAIM) analysis. Our computational calculations revealed that the binding affinity of these compounds is mostly related to the amino‐2H‐imidazole core, which interact tightly with the aspartate dyad of the active site. The interactions were stronger when the inhibitors presented a bulky substituent with a hydrogen bond acceptor motif pointing toward Trp76, such as the 3,5‐dimethyl‐4‐methoxyphenyl group of compound (S)‐1m. Furthermore, the QTAIM analysis revealed that many hydrophobic interactions complement cooperatively the hydrogen bond which is not present when compound (R)‐1t is bound to the enzyme. The combined QM/MM‐QTAIM analysis allows identifying the interactions that account for the activity difference between compounds, even at a nanomolar range.     

Calculation of relative binding affinities of fructose 1,6-bisphosphatase mutants with adenosine monophosphate using free energy perturbation method

The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this article, the importance of hydrophobic and hydrophilic residues to the binding of adenosine monophosphate (AMP) to the fructose 1,6-bisphosphatase (FBPase), a target enzyme for type-II diabetes, was examined by FEP method. Five mutations were made to the FBPase enzyme with AMP inhibitor bound: 113Tyr --> 113Phe, 31Thr --> 31Ala, 31Thr --> 31Ser, 177Met --> 177Ala, and 30Leu --> 30Phe. These mutations test the strength of hydrogen bonds and van der Waals interactions between the ligand and enzyme. The calculated relative free energies indicated that: 113Tyr and 31Thr play an important role, each via two hydrogen bonds affecting the binding affinity of inhibitor AMP to FBPase, and any changes in these hydrogen bonds due to mutations on the protein will have significant effect on the binding affinity of AMP to FBPase, consistent to experimental results. Also, the free energy calculations clearly show that the hydrophilic interactions are more important than the hydrophobic interactions of the binding pocket of FBPase.     

Binding of alkaloids into the S1 specificity pocket of α-chymotrypsin: evidence from induced circular dichroism spectra

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease α-chymotrypsin (αCHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, αCHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which αCHT binding has never been reported. Non-degenerate exciton coupling between π-π* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong αCHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (K(a) ≈ 10(5) M(-1)) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K(a) ≈ 10(4) M(-1)). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of αCHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthermore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of αCHT and related proteases.     

Structural basis for the highly selective inhibition of MMP-13

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of cancer, arthritis, and cardiovascular disease. Here, we report a class of highly selective MMP-13 inhibitors (pyrimidine dicarboxamides) that exhibit no detectable activity against other MMPs. The high-resolution X-ray structures of three molecules of this series bound to MMP-13 reveal a novel binding mode characterized by the absence of interactions between the inhibitors and the catalytic zinc. The inhibitors bind in the S1' pocket and extend into an additional S1' side pocket, which is unique to MMP-13. We analyze the determinants for selectivity and describe the rational design of improved compounds with low nanomolar affinity.     

Hydroxamate类抑制剂与MMP-2的分子对接研究

通过分子动力学模拟研究了MMP-2和hydroxamate抑制剂之间的作用模式。在分子动力学模拟中,对于催化区的锌离子和其共价结合的配体(包括抑制剂和组氨酸)采用了键合的模型。从模拟的结果可以看到,R^1取代基和MMP-2的S1疏水口袋中的部分残基能形成很好的几何匹配,从而可以产生很强的范德华和疏水相互作用。模拟结果也表明,两个抑制剂和MMP-2之间分别能形成5个和8个氢键,抑制剂B比A活性更高的原因就是能够形成更加有利氢键作用模式。在整个模拟过程中,催化锌都能保持好的五配位形式,配位键的长度也处于稳定的状态,预测得到的MMP-2和其抑制剂的相互作用模式对于全新抑制剂的设计提供了非常重要的结构信息。     

Probing the effect of the amidinium group and the phenyl ring on the thermodynamics of binding of benzamidinium chloride to trypsin

The effect of the amidinium group and the phenyl ring on the thermodynamics of binding of benzamidinium chloride to the serine proteinase trypsin has been studied using isothermal titration calorimetry. Binding studies with benzylammonium chloride, [small alpha]-methylbenzylammonium chloride and benzamide, compounds structurally related to benzamidinium chloride, showed that hydrogen bonding between the amidinium group and the enzyme is primarily enthalpy-driven. Binding of cyclohexylcarboxamidinium chloride and acetamidinium chloride showed that the hydrophobic binding of the phenyl ring in the S1 pocket is primarily entropy-driven and that a rigid, flat hydrophobic binding site for the inhibitor is favourable. The compounds that have been studied over a range of temperatures exhibit a negative change in heat capacity upon binding and enthalpy-entropy compensation, both characteristic of hydrophobic interactions.     

Molecular recognition at the active site of factor Xa: cation-π interactions, stacking on planar peptide surfaces, and replacement of structural water

Factor Xa, a serine protease from the blood coagulation cascade, is an ideal enzyme for molecular recognition studies, as its active site is highly shape-persistent and features distinct, concave sub-pockets. We developed a family of non-peptidic, small-molecule inhibitors with a central tricyclic core orienting a neutral heterocyclic substituent into the S1 pocket and a quaternary ammonium ion into the aromatic box in the S4 pocket. The substituents were systematically varied to investigate cation-π interactions in the S4 pocket, optimal heterocyclic stacking on the flat peptide walls lining the S1 pocket, and potential water replacements in both the S1 and the S4 pockets. Structure-activity relationships were established to reveal and quantify contributions to the binding free enthalpy, resulting from single-atom replacements or positional changes in the ligands. A series of high-affinity ligands with inhibitory constants down to K(i)=2 nM were obtained and their proposed binding geometries confirmed by X-ray co-crystal structures of protein-ligand complexes.     

Computational and conformational evaluation of FTase alternative substrates: insight into a novel enzyme binding pocket

Protein farnesyltransferase (FTase) is an important anticancer drug target. In an effort to develop isoprenoid diphosphate-based FTase inhibitors, striking variations have been observed in the ability of conservatively modified analogues to bind to the enzyme. For example, 2Z-GGPP is an alternative substrate with high binding affinity, while GGPP is not an alternative substrate. Using the availability of high-resolution FTase crystal structures, we have used pharmacophore and docking studies to elucidate a new binding pocket for isoprenoid analogues. The unique conformations between the first two isoprene units of 2Z-GGPP, but not GGPP, allows 2Z-GGPP to exploit this new binding pocket. The discovered conformation allows the molecule to adopt a reactive conformation while placing hydrophobic groups within the predominately hydrophobic binding pocket. This computational finding is supported by NMR studies on (13)C-labeled 2Z-farnesol, which confirm that the computationally predicted conformation is also favored in solution. These discoveries suggest that ligand conformational flexibility may be an important design consideration for the development of both inhibitors and alternative substrates of FTase.     

Short communication for targeting natural compounds against HER2 kinase domain as potential anticancer drugs applying pharmacophore based molecular modelling approaches- part 2

Breast cancer is one of the common causes of death noticed in women globally. In order to find effective therapeutics, the current investigation has focussed on identifying candidate compounds for EGFR and HER2. Accordingly, the pharmacophore modelling approaches were adapted to identify two prospective compounds and were docked against the target 3RCD that is complexed with TAK-285 a known dual inhibitor. Focussing on the target 3RCD, our results have showed that the compounds have demonstrated a good binding affinity towards the target occupying the binding pocket. They have established key residue interactions with stable molecular dynamics simulation results. The Hit compounds have demonstrated a potential to penetrate the blood brain barrier thereby enriching their therapeutics towards breast cancer brain metastasis. Taken together, our findings propose two candidate compounds as EGFR/HER2 inhibitors that might serve as novel chemical spaces for designing and developing new inhibitors.     

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.     

Rational design of diflunisal analogues with reduced affinity for human serum albumin.

Many lead compounds bind to serum albumin and exhibit markedly reduced efficacy in vivo as compared to their potency in vitro. To aid in the design of compounds with reduced albumin binding, we performed nuclear magnetic resonance (NMR) structural and binding studies on the complex between domain III of human serum albumin (HSA-III) and diflunisal, a cyclooxygenase inhibitor with antiinflammatory activity. The structural studies indicate that the aromatic rings of diflunisal are involved in extensive and specific interactions with hydrophobic residues that comprise the binding pocket in subdomain IIIA. The carboxylic acid of diflunisal forms electrostatic interactions with the protein similar to those observed in the X-ray structure of HSA complexed to myristic acid. In addition to the structural studies, NMR-derived binding constants were obtained for diflunisal and closely related analogues to develop a structure-affinity relationship for binding to subdomain IIIA. On the basis of the structural and binding data, compounds were synthesized that exhibit more than a 100-fold reduction in binding to domain III of HSA, and nearly a 10-fold reduction in affinity for full length albumin. Significantly, several of these compounds maintain activity against cyclooxygenase-2. These results suggest a rational strategy for designing out albumin binding in potential drug molecules by using structure-based design in conjunction with NMR-based screening.     

Structural Characterization of a Macrocyclic Peptide Modulator of the PD-1/PD-L1 Immune Checkpoint Axis

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.     

Development of a New Class of Inhibitors for the Malarial Aspartic Protease Plasmepsin II Based on a Central 7‐Azabicyclo[2.2.1]heptane Scaffold

Plasmepsin II (PMII), a malarial aspartic protease involved in the catabolism of hemoglobin in parasites of the genus Plasmodium, and renin, a human aspartic protease, share 35% sequence identity in their mature chains. Structures of 4‐arylpiperidine inhibitors complexed to human renin were reported by Roche recently. The major conformational changes, compared to a structure of renin, with a peptidomimetic inhibitor were identified and subsequently modeled in a structure of PMII (Fig. 1). This distorted structure of PMII served as active‐site model for a novel class of PMII inhibitors, according to a structure‐based de novo design approach (Fig. 2). These newly designed inhibitors feature a rigid 7‐azabicyclo[2.2.1]heptane scaffold, which, in its protonated form, is assumed to undergo ionic H‐bonding with the two catalytic Asp residues at the active site of PMII. Two substituents depart from the scaffold for occupancy of either the S1/S3 or S2′‐pocket and the hydrophobic flap pocket, newly created by the conformational changes in PMII. The inhibitors synthesized starting from N‐Boc‐protected 7‐azabicyclo[2.2.1]hept‐2‐ene ( 6 ; Schemes 1–5) displayed up to single‐digit micromolar activity (IC₅₀ values) toward PMII and good selectivity towards renin. The clear structure? activity relationship (SAR; Table) provides strong validation of the proposed conformational changes in PMII and the occupancy of the resulting hydrophobic flap pocket by our new inhibitors.     

Computational Characterization of Binding of Small Molecule Inhibitors to HIV‐1 gp41

Developing orally available small molecule inhibitors of HIV‐1 fusion has attracted significant interest over many years. Frey had recently reported several synthetic compounds which are experimentally shown to inhibit cell‐cell fusion in the low micromolar range. We carried out computational study to help identify possible binding modes by docking these compounds onto the hydrophobic pocket on gp41 and to characterize structures of binding complexes. The detailed gp41‐molecule binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM‐PBSA calculation. Specific molecular interactions in the gp41‐inhibitor complexes are identified. The present computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of small molecular gp41 inhibitors.     

Structure-based design and optimization of antihypertensive peptides to obtain high inhibitory potency against both renin and angiotensin I-converting enzyme

The human renin–angiotensin system (RAS) plays an essential role in regulating blood pressure and systemic vascular resistance. Renin and angiotensin I-converting enzyme (ACE) are two key enzymes in RAS and have long been recognized as attractive antihypertensive targets. Here, a synthetic strategy was proposed integrating quantitative structure–activity relationship (QSAR), molecular dynamics (MD) simulation and binding free energy analysis to discover novel dual renin and ACE peptidic inhibitors. With the strategy a number of candidates were generated virtually, from which eight promising peptides were selected and synthesized for biological assay. Consequently, three peptides (RYLP, YTAWVP and YRAWVL) were successfully identified to have satisfactory inhibitory profile against both renin and ACE with IC₅₀ values of <1 mM and <10 μM, respectively. Structural analysis and energetic dissection revealed different binding modes of peptide to renin and ACE; a peptide only inserts its C-terminus into the active site of ACE, whereas the whole peptide packs tightly against renin. In addition, when limited to structural diversity it is hard to reconcile the renin and ACE inhibitory activities of short peptides such as dipeptides. These findings can be used to guide peptide optimization with improved biological activity.     

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (K_D) for high affinity ligands investigated by nanoESI-MS ranges from 15?nM to 450?nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in K_D and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the K_D values ranging from 2.9 to 17.6???M, some 1?C2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (K_D) measured via nanoESI-MS were compared with kinetic inhibition constants (K_i) in solution.     

Modeling of enzyme–substrate complexes for the metalloproteases MMP-3, ADAM-9 and ADAM-10

The matrix metalloproteases (MMPs) and the ADAMs (A Disintegrin And Metalloprotease domain) are proteolytic enzyme families containing a catalytic zinc ion, that are implicated in a variety of normal and pathological processes involving tissue remodeling and cancer. Synthetic MMP inhibitors have been designed for applications in pathological situations. However, a greater understanding of substrate binding and the catalytic mechanism is required so that more effective and selective inhibitors may be developed for both experimental and clinical purposes. By modeling a natural substrate spanning P4-P4' in complex with the catalytic domains, we aim to compare substrate-specificities between Stromelysin-1 (MMP-3), ADAM-9 and ADAM-10, with the aid of molecular dynamics simulations. Our results show that the substrate retains a favourable antiparallel beta-sheet conformation on the P-side in addition to the well-known orientation of the P'-region of the scissile bond, and that the primary substrate selectivity is dominated by the sidechains in the S1' pocket and the S2/S3 region. ADAM-9 has a hydrophobic residue as the central determinant in the S1' pocket, while ADAM-10 has an amphiphilic residue, which suggests a different primary specificity. The S2/S3 pocket is largely hydrophobic in all three enzymes. Inspired by our molecular dynamics calculations and supported by a large body of literature, we propose a novel, hypothetical, catalytic mechanism where the Zn-ion polarizes the oxygens from the catalytic glutamate to form a nucleophile, leading to a tetrahedral oxyanion anhydride transition state.     

Structure‐Based Design of Nonpeptidic Thrombin Inhibitors: Exploring the D‐Pocket and the Oxyanion Hole

Structure‐activity relationships for new members of a class of nonpeptidic, low‐molecular‐weight inhibitors of thrombin, a key serine protease in the blood coagulation cascade, are described. These compounds, which originate from X‐ray‐structure‐based design, feature a conformationally rigid, bi‐ or tricyclic core from which side chains diverge into the four major binding pockets (distal D, proximal P, recognition or specificity S1, and oxyanion hole O) at the thrombin active site (Fig. 1). Phenylamidinium is the side chain of choice for the S1 pocket, while the most active inhibitors orient an i‐Pr group into the P‐pocket (Table 1). The key step in the synthesis of the inhibitors is the construction of the central bi‐ or tricyclic scaffold by 1,3‐dipolar cycloaddition of an in situ prepared azomethine ylide and an N‐substituted maleimide (Schemes 1–3, and 8–10). One series of compounds was designed to explore the binding features of the large hydrophobic D pocket. This pocket provides space for lipophilic residues as bulky as benzhydryl groups. A new strategy was developed, allowing introduction of these sterically demanding substituents very late in the synthesis (Schemes 5 and 6). Benzhydryl derivative (±)‐ 2 was found to be the most selective member (K_i (trypsin)/K_i (thrombin)=1200) of this class of nonpeptidic thrombin inhibitors, while the ‘dipiperonyl' analog (±)‐ 3 (K_i=9 nM , 7.60‐fold selectivity) displays the highest potency of all compounds prepared so far (Table 1). A second series of inhibitors features side chains designed to orient into the oxyanion hole and to undergo H‐bonding with the backbone NH groups lining the catalytic site of the enzyme. Unfortunately, neither activity nor selectivity could be substantially improved by introduction of these substituents (Table 2). Presumably, the high degree of pre‐organization and the rigidity of the tightly bound scaffolds prevents the new substituents from assuming a position that would allow favorable interactions in the oxyanion hole. However, the oxyanion hole and the S1′ pocket next to it were found to be capable of accommodating quite large groups, which leaves much room for further exploration.