Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.     

DUckCov: a Dynamic Undocking-Based Virtual Screening Protocol for Covalent Binders

Thanks to recent guidelines, the design of safe and effective covalent drugs has gained significant interest. Other than targeting non-conserved nucleophilic residues, optimizing the noncovalent binding framework is important to improve potency and selectivity of covalent binders toward the desired target. Significant efforts have been made in extending the computational toolkits to include a covalent mechanism of protein targeting, like in the development of covalent docking methods for binding mode prediction. To highlight the value of the noncovalent complex in the covalent binding process, here we describe a new protocol using tethered and constrained docking in combination with Dynamic Undocking (DUck) as a tool to privilege strong protein binders for the identification of novel covalent inhibitors. At the end of the protocol, dedicated covalent docking methods were used to rank and select the virtual hits based on the predicted binding mode. By validating the method on JAK3 and KRas, we demonstrate how this fast iterative protocol can be applied to explore a wide chemical space and identify potent targeted covalent inhibitors.     

Ca(2+)-induced conformational transitions of phosphorylated peptides

CD spectroscopic studies on protected peptides containing lysine and serine, or phosphoserine, and on serine-containing fragments of the neurofilament protein midsized subunit, both in the unphosphorylated and phosphorylated form, are reported. The introduction of the phosphoryl group was not found to have a significant spectral effect in aqueous solution. In trifluoroethanol (TFE), spectral shifts toward unordered (type U) spectra or the appearance of distorted spectra likely reflect the adoption of aperiodic polypeptide conformations due to salt bridge(s) between negatively charged phosphoserine and positive lysine side-chain groups. A turn-stabilizing effect of phosphorylation was also observed. CD-monitored titration experiments in TFE revealed a high conformational sensitivity of phosphopeptides toward Ca2+ ions. The appearance of the unordered spectra or spectral shifts were the sign of a bulk disordering effect of Ca2+ ions. Spectra with specific spectroscopic features reflect the formation of Ca2+ complexes and the adoption of ordered unique backbone conformations. When ordered structures were obtained on addition of Ca2+ ions, the observed CD curves showed a resemblance to the spectrum of beta-pleated sheets. This may originate from chain extension and the formation of beta-pleated sheet segments fixed by Ca2+ bridges between PO3H-1 groups of adjacent peptide chains. The data clearly show that the effect of the Ca2+ ions is highly specific: the sequence, chain length, presence and distribution of charged side-chain groups, degree and site of phosphorylation, and environmental factors appear to be determining in the process of chain extension or beta-sheet formation.     

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.     

Compactness of Protein Folds Alters Disulfide-Bond Reducibility by Three Orders of Magnitude: A Comprehensive Kinetic Case Study on the Reduction of Differently Sized Tryptophan Cage Model Proteins

A new approach to monitor disulfide-bond reduction in the vicinity of aromatic cluster(s) has been derived by using the near-UV range (λ=266–293 nm) of electronic circular dichroism (ECD) spectra. By combining the results from NMR and ECD spectroscopy, the 3D fold characteristics and associated reduction rate constants (k) of E19_SS, which is a highly thermostable, disulfide-bond reinforced 39-amino acid long exenatide mimetic, and its N-terminally truncated derivatives have been determined under different experimental conditions. Single disulfide bond reduction of the E19_SS model (with an 18-fold excess of tris(2-carboxyethyl)phosphine, pH 7, 37 °C) takes hours, which is 20–30 times longer than that expected, and thus, would not reach completion by applying commonly used reduction protocols. It is found that structural, steric, and electrostatic factors influence the reduction rate, resulting in orders of magnitude differences in reduction half-lives (900>t_1/2>1 min) even for structurally similar, well-folded derivatives of a small model protein.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins

A new algorithm, called convex constraint analysis, has beendeveloped to deduce the chiral contribution of the common secondarystructures directly from experimental CD curves of a large numberof proteins. The analysis is based on CD data reported by YangJ.T., Wu,C.-S.C. and Martinez,H.M. [Methods Enzymol., 130, 208–269(1986)]. Application of the decomposition algorithm for simulatedprotein data sets resulted in component spectra [B(, i)] identicalto the originals and weights [C(i, k)] with excellent Pearsoncorrelation coefficients (R) [Chang,C.T., Wu,C.-S.C. and Yang,J.T.(1978) Anal. Biochem., 91,12–31]. Test runs were performedon sets of simulated protein spectra created by the Monte Carlotechnique using poly-L-lysine-based pure component spectra.The significant correlational coefficients (R >0.9) demonstratedthe high power of the algorithm. The algorithm, applied to globularprotein data, independent of X-ray data, revealed that the CDspectrum of a given protein is composed of at least four independentsources of chirality. Three of the computed component curvesshow remarkable resemblance to the CD spectra of known proteinsecondary structures. This approach yields a significant improvementin secondary structural evaluations when compared with previousmethods, as compared with X-ray data, and yields a realisticset of pure component spectra. The new method is a useful toolnot only in analyzing CD spectra of globular proteins but alsohas the potential for the analysis of integral membrane proteins.