What We Learned in 25 Years of Interactive Molecular Design Sessions.

We retrace Prof. François Diederich's consultancy work for Roche and its impact over the years he worked with us. François Diederich uniquely shaped our approach to molecular design, and interactions with him and his research group at ETH Zurich have created deep insights into molecular recognition. Herein we share how his style and approach continue to inspire us.

Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group.

This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease-staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.

Synthesis and structure-activity relationship of nitrile-based cruzain inhibitors incorporating a trifluoroethylamine-based P2 amide replacement.

The structure-activity relationship for nitrile-based cruzain inhibitors incorporating a P2 amide replacement based on trifluoroethylamine was explored by deconstruction of a published series of inhibitors. It was demonstrated that the P3 biphenyl substituent present in the published inhibitor structures could be truncated to phenyl with only a small loss of affinity. The effects of inverting the configuration of the P2 amide replacement and linking a benzyl substituent at P1 were observed to be strongly nonadditive. We show that plotting affinity against molecular size provides a means to visualize both the molecular size efficiency of structural transformations and the nonadditivity in the structure-activity relationship. We also show how the relationship between affinity and lipophilicity, measured by high-performance liquid chromatography with an immobilized artificial membrane stationary phase, may be used to normalize affinity with respect to lipophilicity.

Synthesis and structure-activity relationship studies of cruzain and rhodesain inhibitors.

Chagas disease and Human African trypanosomiasis (HAT) are important public health issues in Latin American and sub-Saharan African countries, respectively, and are responsible for a significant number of deaths. The drugs currently used to treat Chagas disease and HAT present efficacy, toxicity, and/or resistance issues; thus, there is a clear need for the discovery of novel targets and drug candidates to combat these diseases. In recent years, much effort has been made to find inhibitors of cruzain and rhodesain, which are promising targets for the design of novel trypanocidal compounds, since they are essential for parasite survival. Many reviews covering the design of novel cruzain and rhodesain inhibitors have been published; however, none have focused on the chemistry of the inhibitors. Thus, in the present work we reviewed the synthetic strategies and routes for the preparation of relevant classes of cruzain and rhodesain inhibitors. Perhaps the most important are the vinyl sulfone derivatives, and a very efficient synthetic strategy based on the Horner-Wadsworth-Emmons reaction was developed to yield these compounds. Modern approaches such as the asymmetric addition of substituted ethynyllithium to N-sulfinyl ketimines were used to produce the chiral alkynes that were employed in the preparation of important chiral triazole derivatives (potent cruzain inhibitors) and chiral HPLC resolution was used for the preparation of enantiopure 3-bromoisoxazoline derivatives (rhodesain inhibitors). Moreover, we also highlight the most important activity results and updated SAR results.

Ultrafiltration, a useful method for isolation of intermediates in native chemical ligation exemplified with the total synthesis of Sortase AΔN59.

In this paper, ultrafiltration was employed to facilitate the isolation of intermediates in native chemical ligation. Depending on the molecular weight cutoff of the membrane used, molecules with different sizes could be purified, separated, or concentrated by the ultrafiltration process. Total chemical synthesis of the polypeptide chain of the enzyme Sortase AΔN59 was used as an example of the application of ultrafiltration in chemical protein synthesis. Sortase A is a ligase that catalyzes transpeptidation reactions between proteins that have C-terminal LPXTG recognition sequence and Gly5- on the peptidoglycan of bacterial cell walls. Ultrafiltration technique facilitated synthesis of Sortase AΔN59 and was a promising tool in isolation of intermediates in native chemical ligation.

Total chemical synthesis of the enzyme sortase A(ΔN59) with full catalytic activity.

The enzyme sortase A is a ligase which catalyzes transpeptidation reactions.1, 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly5 on the peptidoglycan of bacterial cell walls by sortase A.1 The enzyme is an important anti-virulence and anti-infective drug target for resistant strains of Gram-positive bacteria.2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science.3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase AΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size-exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.

Detection of the Sm31 antigen in sera of Schistosoma mansoni- infected patients from a low endemic area.

Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14-18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113-122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83.3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis.

Discovery of selective and nonpeptidic cathepsin S inhibitors.

Nonpeptidic, selective, and potent cathepsin S inhibitors were derived from an in-house pyrrolopyrimidine cathepsin K inhibitor by modification of the P2 and P3 moieties. The pyrrolopyrimidine-based inhibitors show nanomolar inhibition of cathepsin S with over 100-fold selectivity against other cysteine proteases, including cathepsin K and L. Some of the inhibitors showed cellular activities in mouse splenocytes as well as oral bioavailabilities in rats.

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis.

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between naïve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.

Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease.

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.