Druglike, 18F-labeled PET Tracers Targeting Fibroblast Activation Protein.

Fibroblast activation protein (FAP) is a very reliable biomarker for tissue remodeling. FAP has so far mainly been studied in oncology, but there is growing interest in the enzyme in other diseases like fibrosis. Recently, FAP-targeting diagnostics and therapeutics have emerged, of which the so-called FAPIs are among the most promising representatives. FAPIs typically have a relatively high molecular weight and contain very polar, multicharged chelator moieties. While this is not limiting the application of FAPIs in oncology, more druglike FAPIs could be required to optimally study diseases characterized by denser, less permeable tissue. In response, we designed the first druglike 18F-labeled FAPIs. We report target potencies, biodistribution, and pharmacokinetics and demonstrate FAP-dependent uptake in murine tumor xenografts. Finally, this paper puts forward compound 10 as a highly promising, druglike FAPI for 18F-PET imaging. This molecule is fit for additional studies in fibrosis and its preclinical profile warrants clinical investigation.

Design, synthesis and stepwise optimization of nitrile-based inhibitors of cathepsins B and L.

Human cathepsin B (CatB) is an important biological target in cancer therapy. In this work, we performed a knowledge-based design approach and the synthesis of a new set of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over CatK and CatL. The selectivity was achieved by using the combination of a para biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1. Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of enzymes studied. We have also described a successful example of bioisosteric replacement of the amide bond for a sulfonamide one [7e â†’ 6b], where we observed an increase in affinity and selectivity for CatB while lowering the compound lipophilicity (ilogP). Our knowledge-based design approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cathepsins.

Molecular design aided by random forests and synthesis of potent trypanocidal agents as cruzain inhibitors for Chagas disease treatment.

Cruzain is an established target for the identification of novel trypanocidal agents, but how good are in vitro/in vivo correlations? This work describes the development of a random forests model for the prediction of the bioavailability of cruzain inhibitors that are Trypanosoma cruzi killers. Some common properties that characterize drug-likeness are poorly represented in many established cruzain inhibitors. This correlates with the evidence that many high-affinity cruzain inhibitors are not trypanocidal agents against T. cruzi. On the other hand, T. cruzi killers that present typical drug-like characteristics are likely to show better trypanocidal action than those without such features. The random forests model was not outperformed by other machine learning methods (such as artificial neural networks and support vector machines), and it was validated with the synthesis of two new trypanocidal agents. Specifically, we report a new lead compound, Neq0565, which was tested on T. cruzi Tulahuen (ß-galactosidase) with a pEC50 of 4.9. It is inactive in the host cell line showing a selectivity index (SI = EC50cyto /EC50T. cruzi ) higher than 50.

Crystal structure of Leishmania mexicana cysteine protease B in complex with a high-affinity azadipeptide nitrile inhibitor.

Leishmania mexicana is an obligate intracellular protozoan parasite that causes the cutaneous form of leishmaniasis affecting South America and Mexico. The cysteine protease LmCPB is essential for the virulence of the parasite and therefore, it is an appealing target for antiparasitic therapy. A library of nitrile-based cysteine protease inhibitors was screened against LmCPB to develop a treatment of cutaneous leishmaniasis. Several compounds are sufficiently high-affinity LmCPB inhibitors to serve both as starting points for drug discovery projects and as probes for target validation. A 1.4 Å X ray crystal structure, the first to be reported for LmCPB, was determined for the complex of this enzyme covalently bound to an azadipeptide nitrile ligand. Mapping the structure-activity relationships for LmCPB inhibition revealed superadditive effects for two pairs of structural transformations. Therefore, this work advances our understanding of azadipeptidyl and dipeptidyl nitrile structure-activity relationships for LmCPB structure-based inhibitor design. We also tested the same series of inhibitors on related cysteine proteases cathepsin L and Trypanosoma cruzi cruzain. The modulation of these mammalian and protozoan proteases represents a new framework for targeting papain-like cysteine proteases.

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization.

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4'-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

Optimization strategy of single-digit nanomolar cross-class inhibitors of mammalian and protozoa cysteine proteases.

Cysteine proteases (CPs) are involved in a myriad of actions that include not only protein degradation, but also play an essential biological role in infectious and systemic diseases such as cancer. CPs also act as biomarkers and can be reached by active-based probes for diagnostic and mechanistic purposes that are critical in health and disease. In this paper, we present the modulation of a CP panel of parasites and mammals (Trypanosoma cruzi cruzain, LmCPB, CatK, CatL and CatS), whose inhibition by nitrile peptidomimetics allowed the identification of specificity and selectivity for a given CP. The activity cliffs identified at the CP inhibition level are useful for retrieving trends through multiple structure-activity relationships. For two of the cruzain inhibitors (10g and 4e), both enthalpy and entropy are favourable to Gibbs binding energy, thus overcoming enthalpy-entropy compensation (EEC). Group contribution of individual molecular modification through changes in enthalpy and entropy results in a separate partition on the relative differences of Gibbs binding energy (ΔΔG). Overall, this study highlights the role of CPs in polypharmacology and multi-target screening, which represents an imperative trend in the actual drug discovery effort.

Mapping the S1 and S1' subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents.

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. A series of 26 new compounds were designed, synthesized, and tested against the recombinant cruzain (Cz) to map its S1/S1´ subsites. The same series was evaluated on a panel of four human cysteine proteases (CatB, CatK, CatL, CatS) and Leishmania mexicana CPB, which is a potential target for the treatment of cutaneous leishmaniasis. The synthesized compounds are dipeptidyl nitriles designed based on the most promising combinations of different moieties in P1 (ten), P2 (six), and P3 (four different building blocks). Eight compounds exhibited a Ki smaller than 20.0 nM for Cz, whereas three compounds met these criteria for LmCPB. Three inhibitors had an EC50 value of ca. 4.0 µM, thus being equipotent to benznidazole according to the antitrypanosomal effects. Our mapping approach and the respective structure-activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cysteine proteases.

Assessment of the Cruzain Cysteine Protease Reversible and Irreversible Covalent Inhibition Mechanism.

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi (T. cruzi) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor (Neq0682) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682. The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to -26.7 and -5.9 kcal mol-1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the ΔG of the reaction is very negative for the process involving K777, consequently, the covalent adduct cannot revert to a noncovalent protein-ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases.

On the intrinsic reactivity of highly potent trypanocidal cruzain inhibitors.

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known as warhead) are subject to continuous studies to discover novel antichagasic compounds. Here, we evaluated how different warheads for a set of structurally similar related compounds could inhibit the activity of cruzipain and, ultimately, their trypanocidal effect. We first investigated in silico the intrinsic reactivity of these compounds by applying the Fukui index to correlate it with the enzymatic affinity. Then, we evaluated their potency against T. cruzi (Y and Tulahuen strains), which revealed the reversible cruzain inhibitor Neq0656 as a better trypanocidal agent (ECY.strain 50 = 0.1 µM; SI = 58.4) than the current drug benznidazole (ECY.strain 50 = 5.1 µM; SI > 19.6). We also measured the half-life time by HPLC analysis of three lead compounds in the presence of glutathione and cysteine to experimentally assess their intrinsic reactivity. Results clearly illustrated the reactivity trend for the warheads (azanitrile > aldehyde > nitrile), where the aldehyde displayed an intermediate intrinsic reactivity. Therefore, the aldehyde bearing peptidomimetic compounds should be subject for in-depth evaluation in the drug discovery process.

Predicting the affinity of halogenated reversible covalent inhibitors through relative binding free energy.

Nitrile reversible covalent inhibitors of human cathepsin L (hCatL) bind covalently to the side chain of the catalytic Cys25 residue in the S1 pocket to form thioimidates. Predicting the binding of reversible covalent inhibitors is essential for their practical application in drug design. In this report, five nitrile-based inhibitors coded Neq0570, Neq0710, Neq0802, Neq0803 and Neq0804 had their hCatL inhibition constants, Ki, determined. These analogs of the prototypical Neq0570 are halogenated reversible covalent inhibitors of hCatL, which bear a halogen atom in the meta position of the P3 benzyl ring that can form a halogen bond with the Gly61 of the hCatL. To describe halogen bonding interaction in an inhibitor-hCatL complex, we applied an extra point (EP) of charge to represent the anisotropic distribution of charge on the iodine, bromine and chlorine atoms. Besides, we have used alchemical free energy calculations for evaluating the overall relative binding free energies of these inhibitors using a two-state binding model: noncovalent and covalent bond states. Our results show that free energy perturbation (FEP) can predict the hCatL binding affinities of halogenated reversible covalent inhibitors in close agreement with experiments.

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.

Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity?

Cysteine proteases are important targets for the discovery of novel therapeutics for many human diseases. From parasitic diseases to cancer, cysteine proteases follow a common mechanism, the formation of an encounter complex with subsequent nucleophilic reactivity of the catalytic cysteine thiol group toward the carbonyl carbon of a peptide bond or an electrophilic group of an inhibitor. Modulation of target enzymes occurs preferably by covalent modification, which imposes challenges in balancing cross-reactivity and selectivity. Given the resurgence of irreversible covalent inhibitors, can they impair off-target effects or are reversible covalent inhibitors a better route to selectivity? This Perspective addresses how small molecule inhibitors may achieve selectivity for different cathepsins, cruzain, rhodesain, and falcipain-2. We discuss target- and ligand-based designs emphasizing repurposing inhibitors from one cysteine protease to others.

Experimental study and computational modelling of cruzain cysteine protease inhibition by dipeptidyl nitriles.

Chagas disease affects millions of people in Latin America. This disease is caused by the protozoan parasite Trypanossoma cruzi. The cysteine protease cruzain is a key enzyme for the survival and propagation of this parasite lifecycle. Nitrile-based inhibitors are efficient inhibitors of cruzain that bind by forming a covalent bond with this enzyme. Here, three nitrile-based inhibitors dubbed Neq0409, Neq0410 and Neq0570 were synthesized, and the thermodynamic profile of the bimolecular interaction with cruzain was determined using isothermal titration calorimetry (ITC). The result suggests the inhibition process is enthalpy driven, with a detrimental contribution of entropy. In addition, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to investigate the reaction mechanism of reversible covalent modification of cruzain by Neq0409, Neq0410 and Neq0570. The computed free energy profile shows that the nucleophilic attack of Cys25 on the carbon C1 of inhibitiors and the proton transfer from His162 to N1 of the dipeptidyl nitrile inhibitor take place in a single step. The calculated free energy of the inhibiton reaction is in agreement with covalent experimental binding. Altogether, the results reported here suggests that nitrile-based inhibitors are good candidates for the development of reversible covalent inhibitors of cruzain and other cysteine proteases.

Natural Aromatic Compounds as Scaffolds to Develop Selective G-Quadruplex Ligands: From Previously Reported Berberine Derivatives to New Palmatine Analogues.

In this paper, the selective interactions of synthetic derivatives of two natural compounds, berberine and palmatine, with DNA G-quadruplex structures were reported. In particular, the previous works on this subject concerning berberine were further presented and discussed, whereas the results concerning palmatine are presented here for the first time. In detail, these palmatine derivatives were developed by inserting seven different small peptide basic chains, giving several new compounds that have never been reported before. The preliminary studies of the interactions of these compounds with various G-quadruplex-forming sequences were carried out by means of various structural and biochemical techniques, which showed that the presence of suitable side chains is very useful for improving the interaction of the ligands with G-quadruplex structures. Thus, these new palmatine derivatives might act as potential anticancer drugs.

Leveraging the cruzain S3 subsite to increase affinity for reversible covalent inhibitors.

Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease. Reversible covalent cruzain inhibitors can block the steps of cell differentiation in the parasite and kill the organism. To this end, the description of how inhibitors modified at the P2/P3 positions lead to analogs with greater cruzain affinity to the S2/S3 subsites is of fundamental importance. Albeit many efforts are being employed in the characterization of the interaction processes with S2 subsite, little is known about the cruzain S3 subsite. In this work, we show a brief but consistent study to identify favorable substitutions in P3 of dipeptidyl nitriles that increase cruzain affinity. Using molecular dynamics simulations, we have identified some dipeptidyl nitrile analogs with modifications at P3 position that had higher cruzain inhibition than the original unsubstituted compound. A matched molecular pair analysis shows the importance of including a chlorine atom in the P3-meta position. The modifications implemented in P3 are confirmed when profiling the thermodynamic parameters via isothermal titration calorimetry. The classical enthalpy-entropy compensation phenomenon, in which enthalpy changes are counterbalanced by entropy results in a small modification of ΔG. The inclusion of the chlorine atom in the P3-meta position results in the highest reduction of the detrimental entropic contribution observed in P3.

A comparative study of warheads for design of cysteine protease inhibitors.

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.

Perylene and coronene derivatives binding to G-rich promoter oncogene sequences efficiently reduce their expression in cancer cells.

A novel approach to cancer therapeutics is emerging in the field of G-quadruplex (G4) ligands, small molecules designed to stabilize four-stranded structures that can form at telomeres as well as in other genomic sequences, including oncogene promoter sequences, 5'-UTR regions and introns. In this study, we investigated the binding activity of perylene and coronene derivatives PPL3C, CORON and EMICORON to G4 structures formed within the promoter regions of two important cancer-related genes, c-MYC and BCL-2, and their biochemical effects on gene and protein expression. In order to fully characterize the ability of the selected ligands to bind and stabilize the G4 structures originated by the c-MYC and BCL-2 promoter sequences, we performed electrospray ionization mass spectrometry (ESI-MS), Fluorescence Resonance Energy Transfer (FRET) measurements, Circular Dichroism (CD) spectra and polymerase stop assay. Altogether our results showed that the ligands had a high capacity in binding and stabilizing the G4 structures within the c-MYC and BCL-2 promoter sequences in vitro. Notably, when we evaluated by quantitative real-time PCR and western blotting analysis, the effects of treatment with the different G4 ligands on c-MYC and BCL2 expression in a human melanoma cell line, EMICORON appeared the most effective compound in reducing the mRNA and protein levels of both genes. These results encourage to consider EMICORON as a promising example of multimodal class of an antineoplastic drug, affecting different tumor crucial pathways simultaneously: telomere maintenance (as previously described), cell proliferation and apoptosis via down-regulation of both c-MYC and BCL-2 (this paper).