Rational design of improved aziridine-based inhibitors of cysteine proteases.

Quantum chemical computations on appropriate model systems are used for a rational design of aziridine-based inhibitors of cysteine proteases. They predict that already inductive electron-withdrawing substituents at the aziridine nitrogen strongly accelerate the alkylation step of the inhibition process in neutral and alkaline media, but also for more acidic environments improvements are predicted. With this we generalize previous findings that found similar effects for N-formylated compounds. Furthermore, the new substituents possess the additional advantage that they do not open up reaction pathways other than the nucleophilic ring opening. To verify the hypotheses selected compounds were synthesized and tested. These tests approved the predictions and showed that the corresponding derivatives of aziridine-2,3-dicarboxylate are potent irreversible inhibitors of cysteine proteases. On the basis of measured inhibition data the new inhibitors offer an up to 2,300-fold increase in inhibition potency compared to the unsubstituted inhibitor. Additionally, the kinetics of a selected reaction with 4-methoxy thiophenolate as model thiol were measured in solution to ascertain that the inhibition mechanism is the irreversible alkylation of the cysteine residue of the protease's active site under ring opening of the new inhibitors.

Environmental effects on charge densities of biologically active molecules: do molecule crystal environments indeed approximate protein surroundings?

In the present paper, we investigate whether crystal and enzyme environments influence the electron density (ED) of active compounds in a similar manner. This supposition is essential for high-resolution X-ray studies, which use the EDs obtained from crystals of the pure active compound as approximations for the ED of the active compound in its complex with the target enzyme. The EDs of such complexes determine the molecular recognition process between the targeted enzyme and active compound and are, hence, extremely useful tools for rational drug design. The approximation of such EDs by data obtained from crystals of the pure active compound is needed since high-resolution X-ray experiments of the target-ligand complexes are still extremely demanding. Quantum mechanical/molecular mechanical (QM/MM) and pure QM calculations are employed to determine the EDs of two inhibitors, the reversible trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA) and the irreversible E64c in four different environments (the enzyme-inhibitor complex, crystals of the pure compounds, a continuum solvation model, and the gas phase). Our investigation shows that the environment inside of the crystal of the pure active compound generally influences the ED of an active compound in a very similar way as the enzyme surrounding in the complex between the active compound and target enzyme. However, this does not hold any more if the geometrical arrangement of the inhibitor in the enzyme differs significantly from that in the crystal. While EDs computed for gas-phase environments deviate strongly from those in crystal and protein surroundings, polar solvent environments provide rather similar electron distributions. Thus, such continuum solvation models are very well suited to compute density databases which are to be employed for the determination of the ED of macromolecules.

Atomistic insights into the inhibition of cysteine proteases: first QM/MM calculations clarifying the stereoselectivity of epoxide-based inhibitors.

Due to their important role in many diseases, cysteine proteases represent new promising drug targets. An important class of cysteine-protease inhibitors is derived from the naturally occurring compound E64, possessing an epoxysuccinyl moiety as warhead. Experimental studies show stereoselectivity concerning the inhibition potency, e.g., a trans-configured epoxide ring is essential for inhibition, and furthermore, in most cases, the ( S, S)-configured inhibitors have a higher inhibition potency than their ( R, R)-counterparts. However, the underlying effects are not fully understood. In this work, such effects are investigated by classical molecular dynamics simulations and combined quantum mechanics/molecular modeling (QM/MM) calculations for the E64c-cathepsin B complex. Our computations reveal that the hydrogen bonding network between the enzyme and the E64c (or its derivatives) determines the stereoselectivity of the subsequent ring opening reaction by governing the distance between the attacking thiolate and the attacked C2 atom of the epoxide ring. For the ( S, S)-configuration, a strong network can be realized which enables a close contact between the reacting centers, so that the irreversible step becomes very efficient. The ( R, S)-configuration ( cis-configuration) can only form networks in which the two reacting centers are so far away from each other that the irreversible step can hardly happen. The ( R, R)-configuration is in between, less optimal than the ( S, S)-configuration but much better than the ( R, S)-configuration. Exceptions where the ( R, R)-configurations shows higher potency than the ( S, S) ones are also explained.

On the origin of the stabilization of the zwitterionic resting state of cysteine proteases: a theoretical study.

Papain-like cysteine proteases are ubiquitous proteolytic enzymes. The protonated His199/deprotonated Cys29 ion pair (cathepsin B numbering) in the active site is essential for their proper functioning. The presence of this ion pair stands in contrast to the corresponding intrinsic residue p K a values, indicating a strong influence of the enzyme environment. In the present work we show by molecular dynamics simulations on quantum mechanical/molecular mechanical (QM/MM) potentials that the ion pair is stabilized by a complex hydrogen bond network which comprises several amino acids situated in the active site of the enzyme and 2-4 water molecules. QM/MM reaction path computations for the proton transfer from His199 to the thiolate of the Cys29 moiety indicate that the ion pair is about 32-36 kJ mol (-1) more stable than the neutral form if the whole hydrogen bonding network is active. Without any hydrogen bonding network the ion pair is predicted to be significantly less stable than the neutral form. QM/MM charge deletion analysis and QM model calculations are used to quantify the stabilizing effect of the active-site residues and the L1 helix in favor of the zwitterionic form. The active-site water molecules contribute about 30 kJ mol (-1) to the overall stabilization. Disruption of the hydrogen bonding network upon substrate binding is expected to enhance the nucleophilic reactivity of the thiolate.

Atomistic insights into the inhibition of cysteine proteases: first QM/MM calculations clarifying the regiospecificity and the inhibition potency of epoxide- and aziridine-based inhibitors.

Epoxides and aziridines are important building blocks for inhibitors of cysteine proteases which are promising drug targets for many diseases. In spite of the large amount of experimental data concerning inhibition potency, structure-activity relationships, and structural arrangements of enzyme-inhibitor complexes, little is known about the basic principles which connect the substitution pattern with the resulting activities. To shed some light on this issue which is essential for the rational design of improved compounds, we have studied the inhibition processes theoretically for various inhibitors using quantum mechanical/molecular mechanical hybrid approaches and classical molecular dynamics simulations. The careful analysis of the computational results allows insight into the interactions which govern the regio- and stereospecificity of the interactions. Known structure-activity relationships are rationalized in terms of the same interactions that determine the measured pH dependencies. Inconsistencies in existing X-ray structures are resolved through comparison with the computed structures, which leads to a reassessment of the factors that control the inhibition potency. Similarities and differences in the mode of action of epoxide- and aziridine-based inhibitors are elucidated. Finally the small reaction barriers computed for the irreversible step in E64 analogues call into question the commonly accepted two-step model of inhibition since the second, irreversible step is predicted to be so fast that suitably oriented enzyme-inhibitor complexes will react rather than dissociate and equilibrate.

The importance of the active site histidine for the activity of epoxide- or aziridine-based inhibitors of cysteine proteases.

In the present study the importance of the active site histidine residue (His) for the activity of epoxide- or aziridine-based cysteine protease inhibitors is examined theoretically. To account for all important effects, QM/MM hybrid approaches are employed which combine quantum mechanical (QM) methods that are necessary to describe bond-breaking and formation processes, with molecular mechanics (MM) methods that incorporate the influence of the protein environment. Using various model systems, the computations exclude a direct proton shift from the active site His residue to the inhibitor, but show that one water molecule is sufficient to establish a very efficient relay system. This relay system allows an easy proton transfer from the active site His residue to the inhibitor and is thus essential for the activity of both types of inhibitors. Differences between the epoxides and the aziridines are discussed, along with some implications for the rational design of optimized inhibitors. The work presented herein represents the first QM/MM study into the mode of action of these important inhibitor classes.

Aziridide-based inhibitors of cathepsin L: synthesis, inhibition activity, and docking studies.

A comprehensive screening of N-acylated aziridine (aziridide) based cysteine protease inhibitors containing either Boc-Leu-Caa (Caa=cyclic amino acid), Boc-Gly-Caa, or Boc-Phe-Ala attached to the aziridine nitrogen atom revealed Boc-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)(2) (18 a) as a highly potent cathepsin L (CL) inhibitor (K(i)=13 nM) (Azy=aziridine-2-carboxylate, Azi=aziridine-2,3-dicarboxylate). Docking studies, which also accounted for the unusual bonding situations (the flexibility and hybridization of the aziridides) predict that the inhibitor adopts a Y shape and spans across the entire active site cleft, binding into both the nonprimed and primed sites of CL.

Geometry and cooperativity effects in adenosine-carboxylic acid complexes.

NMR experiments and theoretical investigations were performed on hydrogen bonded complexes of specifically 1- and 7-15N-labeled adenine nucleosides with carboxylic acids. By employing a freonic solvent of CDClF2 and CDF3, NMR spectra were acquired at temperatures as low as 123 K, where the regime of slow hydrogen bond exchange is reached and several higher-order complexes were found to coexist in solution. Unlike acetic acid, chloroacetic acid forms Watson-Crick complexes with the proton largely displaced from oxygen to the nitrogen acceptor in an ion pairing structure. Calculated geometries and chemical shifts of the proton in the hydrogen bridge favorably agree with experimentally determined values if vibrational averaging and solvent effects are taken into account. The results indicate that binding a second acidic ligand at the adenine Hoogsteen site in a ternary complex weakens the hydrogen bond to the Watson-Crick bound carboxylic acid. However, substituting a second adenine nucleobase for a carboxylic acid in the trimolecular complex leads to cooperative binding at Watson-Crick and Hoogsteen faces of adenosine.

An ab initio study of the vibronic, spin-orbit, and magnetic hyperfine structure in the X2Pi electronic state of NCO.

In the present study we give the results of the ab initio calculations on the vibronic, spin-orbit, and magnetic hyperfine structure in the X (2)Pi electronic state of the NCO radical. The calculations of the potential surfaces and the electronic mean values of the hyperfine coupling constants are carried out by means of the density functional theory approach (B3LYP functional combined with an atomic orbital basis set suitable for calculations of the hyperfine structure). The vibronic levels, spin-orbit splitting, and the vibronic mean values of the components of the hyperfine tensor in the vibronic species are calculated using a variational method. The results of the calculations are in good agreement with the available experimental data.

An ab initio calculation of the anisotropic hyperfine coupling constants in the low-lying vibronic levels of the X 2Pi electronic state of CCCH.

The results of ab initio calculations of the vibronically averaged components of the anisotropic magnetic hyperfine tensor in the low-lying vibronic species of the X (2)Pi electronic state of CCCH and CCCD are reported. The electronically averaged hyperfine coupling constants for hydrogen and (13)C in (12)C (12)C (12)CH, (13)C (12)C (12)CH, (12)C (13)C (12)CH, (12)C (12)C (13)CH, and (12)C (12)C (12)CD are obtained as functions of two bending vibrational modes by the density functional theory method. The vibronic wave functions are calculated with help of a variational approach which takes into account the Renner-Teller effect and spin-orbit coupling. The results of the present study help to reliably interpret the experimental data previously published and predict the yet unobserved hyperfine structure in excited vibronic states of CCCH and CCCD.

An ab initio study of the hyperfine structure in the X2 Pi electronic state of CCCH.

The results of an ab initio study of the magnetic hyperfine structure in the X (2)Pi electronic state of CCCH are reported. The potential surfaces for two components of the X (2)Pi electronic state were computed by means of an extensive configuration interaction approach. The electronically averaged hyperfine coupling constants of H and (13)C for (12)C (12)C (12)CH, (13)C (12)C (12)CH, (12)C (13)C (12)CH, and (12)C (12)C (13)CH are obtained as functions of two bending vibrational modes by the density functional theory method. The vibronic wave functions are calculated with the help of a variational approach which takes into account the Renner-Teller effect and spin-orbit coupling. The model Hamiltonian is expressed in terms of the normal bending coordinates. It is found that, due to the generally strong geometry dependence of the hyperfine coupling constants, it is necessary to carry out the vibronic averaging of the corresponding functions in order to obtain the values which can be compared to the results of the measurements. The results of the present study help to reliably interpret the experimental data previously published. They also predict the yet unobserved hyperfine structure in excited vibronic states.