Zika virus replication is impaired by a selective agonist of the TRPML2 ion channel.

The flavivirus genus includes human pathogenic viruses such as Dengue (DENV), West Nile (WNV) and Zika virus (ZIKV) posing a global health threat due to limited treatment options. Host ion channels are crucial for various viral life cycle stages, but their potential as targets for antivirals is often not fully realized due to the lack of selective modulators. Here, we observe that treatment with ML2-SA1, an agonist for the human endolysosomal cation channel TRPML2, impairs ZIKV replication. Upon ML2-SA1 treatment, levels of intracellular genomes and number of released virus particles of two different ZIKV isolates were significantly reduced and cells displayed enlarged vesicular structures and multivesicular bodies with ZIKV envelope protein accumulation. However, no increased ZIKV degradation in lysosomal compartments was observed. Rather, the antiviral effect of ML2-SA1 seemed to manifest by the compound's negative impact on genome replication. Moreover, ML2-SA1 treatment also led to intracellular cholesterol accumulation. ZIKV and many other viruses including the Orthohepevirus Hepatitis E virus (HEV) rely on the endolysosomal system and are affected by intracellular cholesterol levels to complete their life cycle. Since we observed that ML2-SA1 also negatively impacted HEV infections in vitro, this compound may harbor a broader antiviral potential through perturbing the intracellular cholesterol distribution. Besides indicating that TRPML2 may be a promising target for combatting viral infections, we uncover a tentative connection between this protein and cholesterol distribution within the context of infectious diseases.

Structural features and antiproliferative activity of Pd(II) complexes with halogenated ligands: a comparative study between Schiff base and reduced Schiff base complexes.

In order to investigate the structural features and antiproliferative activity of Pd(II) complexes containing halogenated ligands with different flexibility, several Schiff base and reduced Schiff base Pd(II) complexes, namely X1X2PicPd, X1X2PyPd, X1X2Pic(R)Pd, and X1X2Py(R)Pd (where X1 = X2 = Cl, Br and I; Pic: 2-picolylamine; Py = 2-(2-pyridyl)ethylamine), were synthesized and characterized by spectroscopic methods and, in the case of Br2PyPd, Cl2Py(R)Pd and ClBrPy(R)Pd, also by X-ray crystallography. The results of the X-ray crystallography showed that in both series of complexes the Pd(II) ion has a distorted square-planar geometry, although the coordination modes of the two ligands are different. In the Schiff base-type complexes the ligand acts as a tridentate chelate with NN'O donor atoms, whereas in the reduced Schiff base-type complexes the ligand acts as a bidentate chelate with NN' donor atoms. In both series of complexes, the chloride ions occupy the residual coordination sites of the Pd(II) ion. TD-DFT calculations were performed for a better understanding of the UV-Vis spectra. From these calculations it was found that the signal appearing at ∼400 nm in the complexes with reduced Schiff base ligands (X1X2Pic(R)Pd and X1X2Py(R)Pd) is mainly due to a HOMO → LUMO transition, while for the Schiff base complex ClBrPyPd the signal is due to a HOMO → LUMO+1 transition. For the complex I2PicPd, combinations of HOMO-4 → LUMO and HOMO-2 → LUMO transitions were found to be responsible for that signal. In regard to the biological activity profile, all complexes were first investigated as proteasome inhibitors by fluorometric methods. From these enzymatic assays, it emerged that they are good inhibitors with IC50 values in the low-micromolar range and that their inhibitory activity is strictly related to the presence of the metal ion. Subsequently they were also subjected to cell-based assays (the resazurin method) to assess their antiproliferative properties by using two leukemic cell lines, namely the drug-sensitive CCRF-CEM cell line and its multidrug-resistant sub-cell line CEM/ADR5000. In this test they displayed IC50 values in the sub-micromolar and low-micromolar range determined for a selected metal complex (Br2Pic(R)Pd) and ligand (Cl2Pic(R)), respectively. Moreover, docking studies were performed on the two expected molecular targets, i.e. proteasome and DNA, to shed light on the mechanisms of action of these types of Pd(II) complexes.

Structure-Based Design and Synthesis of Covalent Inhibitors for Deubiquitinase and Acetyltransferase ChlaDUB1 of Chlamydia trachomatis.

Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.

Substitution-Induced Mechanistic Switching in SNAr-Warheads for Cysteine Proteases.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Structural Modifications of Covalent Cathepsin S Inhibitors: Impact on Affinity, Selectivity, and Permeability.

Cathepsin S (catS) is a member of the cysteine protease family with limited tissue distribution, which is predominantly found in antigen-presenting cells. Due to overexpression and overactivity of catS in numerous cancers, inhibition of catS is supposed to improve the antitumor response. Here, we explore the potential of small-molecule catS inhibitors emphasizing their in vitro pharmacodynamics and pharmacokinetics. Membrane permeability of selected inhibitors was measured with a Parallel Artificial Membrane Permeation Assay and correlated to calculated physicochemical parameters and inhibition data. The binding kinetics and inhibition types of potent and selective new inhibitors with unexplored warheads were investigated. Our unique approach involves reversible masking of these potent warheads, allowing for further customization without compromising affinity or selectivity. The most promising inhibitors in this study include covalent aldehyde and ketone derivatives reversibly masked as hydrazones as potential candidates for therapeutic interventions targeting catalytic enzymes and modulating the immune response in cancer.

Identification of Dual Inhibitors Targeting Main Protease (Mpro) and Cathepsin L as Potential Anti-SARS-CoV-2 Agents.

In this structure-activity relationship (SAR) study, we report the development of dual inhibitors with antiviral properties targeting the SARS-CoV-2 main protease (Mpro) and human cathepsin L (hCatL). The novel molecules differ in the aliphatic amino acids at the P2 site and the fluorine position on the phenyl ring at the P3 site. The identified dual inhibitors showed Ki values within 1.61 and 10.72 µM against SARS-CoV-2 Mpro; meanwhile, Ki values ranging from 0.004 to 0.701 µM toward hCatL were observed. A great interdependency between the nature of the side chain at the P2 site and the position of the fluorine atom was found. Three dual-targeting inhibitors exhibited antiviral activity in the low micromolar range with CC50 values >100 µM. Docking simulations were executed to gain a deeper understanding of the SAR profile. The findings herein collected should be taken into consideration for the future development of dual SARS-CoV-2 Mpro/hCatL inhibitors.

Development of Novel Peptidyl Nitriles Targeting Rhodesain and Falcipain-2 for the Treatment of Sleeping Sickness and Malaria.

In recent decades, neglected tropical diseases and poverty-related diseases have become a serious health problem worldwide. Among these pathologies, human African trypanosomiasis, and malaria present therapeutic problems due to the onset of resistance, toxicity problems and the limited spectrum of action. In this drug discovery process, rhodesain and falcipain-2, of Trypanosoma brucei rhodesiense and Plasmodium falciparum, are currently considered the most promising targets for the development of novel antitrypanosomal and antiplasmodial agents, respectively. Therefore, in our study we identified a novel lead-like compound, i.e., inhibitor 2b, which we proved to be active against both targets, with a Ki = 5.06 µM towards rhodesain and an IC50 = 40.43 µM against falcipain-2.

Dual Strategy to Design New Agents Targeting Schistosoma mansoni: Advancing Phenotypic and SmCB1 Inhibitors for Improved Efficacy.

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 µM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 µM, 68% at 1 µM, 35% at 0.1 µM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.

Ligand-Based Design of Selective Peptidomimetic uPA and TMPRSS2 Inhibitors with Arg Bioisosteres.

Trypsin-like serine proteases are involved in many important physiological processes like blood coagulation and remodeling of the extracellular matrix. On the other hand, they are also associated with pathological conditions. The urokinase-pwlasminogen activator (uPA), which is involved in tissue remodeling, can increase the metastatic behavior of various cancer types when overexpressed and dysregulated. Another member of this protease class that received attention during the SARS-CoV 2 pandemic is TMPRSS2. It is a transmembrane serine protease, which enables cell entry of the coronavirus by processing its spike protein. A variety of different inhibitors have been published against both proteases. However, the selectivity over other trypsin-like serine proteases remains a major challenge. In the current study, we replaced the arginine moiety at the P1 site of peptidomimetic inhibitors with different bioisosteres. Enzyme inhibition studies revealed that the phenylguanidine moiety in the P1 site led to strong affinity for TMPRSS2, whereas the cyclohexylguanidine derivate potently inhibited uPA. Both inhibitors exhibited high selectivity over other structurally similar and physiologically important proteases.

Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 1-10 µM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1-12 µM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

Investigating the Cytotoxicity of Ru(II) Polypyridyl Complexes by Changing the Electronic Structure of Salicylaldehyde Ligands.

A novel class of Ru(II)-based polypyridyl complexes with an auxiliary salicylaldehyde ligand [Ru(phen)2(X-Sal)]BF4 {X: H (1), 5-Cl (2), 5-Br (3), 3,5-Cl2 (4), 3,5-Br2 (5), 3-Br,5-Cl (6), 3,5-I2 (7), 5-NO2 (8), 5-Me (9), 4-Me (10), 4-OMe (11), and 4-DEA (12), has been synthesized and characterized by elemental analysis, FT-IR, and 1H/13C NMR spectroscopy. The molecular structure of 4, 6, 9, 10, and 11 was determined by single-crystal X-ray diffraction analysis which revealed structural similarities. DFT and TD-DFT calculations showed that they also possess similar electronic structures. Absorption/emission spectra were recorded for 2, 3, 10, and 11. All Ru-complexes, unlike the pure ligands and the complex lacking the salicylaldehyde component, displayed outstanding antiproliferative activity in the screening test (10 µM) against CCRF-CEM leukemia cells underlining the crucial role of the presence of the auxiliary ligand for the biological activity. The two most active derivatives, namely 7 and 10, were selected for continuous assays showing IC50 values in the submicromolar and micromolar range against drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells, respectively. These two compounds were investigated in silico for their potential binding to duplex DNA well-matched and mismatched base pairs, since they showed remarkable selectivity indexes (2.2 and 19.5 respectively) on PBMC cells.

Designing photoaffinity tool compounds for the investigation of the DENV NS2B-NS3 protease allosteric binding pocket.

Dengue virus (DENV) infection still lacks specific antiviral therapy, making the NS2B-NS3 protease an attractive target for drug development. However, allosteric inhibitors that bind to a site other than the active site still need to be better understood. In this study, we designed and synthesised tool compounds for photoaffinity labelling (PAL) to investigate the binding site of allosteric inhibitors on the DENV protease. These tool compounds contained an affinity moiety, a photoreactive group, and a reporter tag for detection. Upon irradiation, the photoreactive group formed a covalent bond with the protease, allowing for binding site identification. SDS-PAGE-based assays confirmed the qualitative binding of the designed inhibitors to the allosteric pocket, and pull-down experiments validated the interaction. Tryptic protein digestion following liquid chromatography/mass spectrometry analysis further supported the binding of the inhibitor to the proposed pocket revealing photo-attachment to an NS3 loop close to the C-terminus. These results enhance our understanding of allosteric inhibitors and their mechanism of action against the DENV protease. The developed tool compounds and PAL are potent tools for future drug discovery efforts and investigations targeting the DENV protease.

Impact of the Warhead of Dipeptidyl Keto Michael Acceptors on the Inhibition Mechanism of Cysteine Protease Cathepsin L.

Cathepsin L (CatL) is a lysosomal cysteine protease whose activity has been related to several human pathologies. However, although preclinical trials using CatL inhibitors were promising, clinical trials have been unsuccessful up to now. We are presenting a study of two designed dipeptidyl keto Michael acceptor potential inhibitors of CatL with either a keto vinyl ester or a keto vinyl sulfone (KVS) warhead. The compounds were synthesized and experimentally assayed in vitro, and their inhibition molecular mechanism was explored based on molecular dynamics simulations at the density functional theory/molecular mechanics level. The results confirm that both compounds inhibit CatL in the nanomolar range and show a time-dependent inhibition. Interestingly, despite both presenting almost equivalent equilibrium constants for the reversible formation of the noncovalent enzyme/inhibitor complex, differences are observed in the chemical step corresponding to the enzyme-inhibitor covalent bond formation, results that are mirrored by the computer simulations. Theoretically determined kinetic and thermodynamic results, which are in very good agreement with the experiments, afford a detailed explanation of the relevance of the different structural features of both compounds having a significant impact on enzyme inhibition. The unprecedented binding interactions of both inhibitors in the P1' site of CatL represent valuable information for the design of inhibitors. In particular, the peptidyl KVS can be used as a starting lead compound in the development of drugs with medical applications for the treatment of cancerous pathologies since sulfone warheads have previously shown promising cell stability compared to other functions such as carboxylic esters. Future improvements can be guided by the atomistic description of the enzyme-inhibitor interactions established along the inhibition reaction derived from computer simulations.

Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells.

The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investigation. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immunofluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents.

Covalent S-Adenosylhomocysteine-Based DNA Methyltransferase 2 Inhibitors with a New Type of Aryl Warhead.

The DNA methyltransferase 2 (DNMT2) is an RNA modifying enzyme associated with pathophysiological processes, such as mental and metabolic disorders or cancer. Although the development of methyltransferase inhibitors remains challenging, DNMT2 is not only a promising target for drug discovery, but also for the development of activity-based probes. Here, we present covalent SAH-based DNMT2 inhibitors decorated with a new type of aryl warhead. Based on a noncovalent DNMT2 inhibitor with N-benzyl substituent, the Topliss scheme was followed for optimization. The results showed that electron-deficient benzyl moieties highly increased affinity. By decorating the structures with strong electron-withdrawing moieties and leaving groups, we adjusted the electrophilicity to create covalent DNMT2 inhibitors. A 4-bromo-3-nitrophenylsulfonamide-decorated SAH derivative (80) turned out to be the most potent (IC50 = 1.2 ± 0.1 µM) and selective inhibitor. Protein mass spectrometry confirmed the covalent reaction with the catalytically active cysteine-79.

Next Generation of Fluorometric Protease Assays: 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-Amides) as Class-Spanning Protease Substrates.

Fluorometric assays are one of the most frequently used methods in medicinal chemistry. Over the last 50 years, the reporter molecules for the detection of protease activity have evolved from first-generation colorimetric p-nitroanilides, through FRET substrates, and 7-amino-4-methyl coumarin (AMC)-based substrates. The aim of further substrate development is to increase sensitivity and reduce vulnerability to assay interferences. Herein, we describe a new generation of substrates for protease assays based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-amides). In this study, we synthesized and tested substrates for 10 different proteases from the serine-, cysteine-, and metalloprotease classes. Enzyme- and substrate-specific parameters as well as the inhibitory activity of literature-known inhibitors confirmed their suitability for application in fluorometric assays. Hence, we were able to present NBD-based alternatives for common protease substrates. In conclusion, these NBD substrates are not only less susceptible to common assay interference, but they are also able to replace FRET-based substrates with the requirement of a prime site amino acid residue.

The effects of allosteric and competitive inhibitors on ZIKV protease conformational dynamics explored through smFRET, nanoDSF, DSF, and 19F NMR.

Zika and dengue viruses cause mosquito-borne diseases of high epidemic relevance. The viral NS2B-NS3 proteases play crucial roles in the pathogen replication cycle and are validated drug targets. They can adopt at least two conformations depending on the position of the NS2B cofactor. Recently, we reported ligand-induced conformational changes of dengue virus NS2B-NS3 protease by single-molecule Förster resonance energy transfer (smFRET). Here, we investigated the conformational dynamics of the homologous Zika virus protease through an integrated methodological approach combining smFRET, thermal shift assays (DSF and nanoDSF) and 19F NMR spectroscopy. Our results show that allosteric inhibitors favor the open conformation and competitive inhibitors stabilize the closed conformation of the Zika virus protease.

Impact of the Recognition Part of Dipeptidyl Nitroalkene Compounds on the Inhibition Mechanism of Cysteine Proteases Cruzain and Cathepsin L.

Cysteine proteases (CPs) are an important class of enzymes, many of which are responsible for several human diseases. For instance, cruzain of protozoan parasite Trypanosoma cruzi is responsible for the Chagas disease, while the role of human cathepsin L is associated with some cancers or is a potential target for the treatment of COVID-19. However, despite paramount work carried out during the past years, the compounds that have been proposed so far show limited inhibitory action against these enzymes. We present a study of proposed covalent inhibitors of these two CPs, cruzain and cathepsin L, based on the design, synthesis, kinetic measurements, and QM/MM computational simulations on dipeptidyl nitroalkene compounds. The experimentally determined inhibition data, together with the analysis and the predicted inhibition constants derived from the free energy landscape of the full inhibition process, allowed describing the impact of the recognition part of these compounds and, in particular, the modifications on the P2 site. The designed compounds and, in particular, the one with a bulky group (Trp) at the P2 site show promising in vitro inhibition activities against cruzain and cathepsin L for use as a starting lead compound in the development of drugs with medical applications for the treatment of human diseases and future designs.

Influence of amino acid size at the P3 position of N-Cbz-tripeptide Michael acceptors targeting falcipain-2 and rhodesain for the treatment of malaria and human african trypanosomiasis.

In recent decades, several structure-activity relationship (SAR) studies provided potent inhibitors of the cysteine proteases falcipain-2 (FP-2) and rhodesain (RD) from Plasmodium falciparum and Trypanosoma brucei rhodesiense, respectively. Whilst the roles of the warhead and residues targeting the P1 and P2 pockets of the proteases were extensively investigated, the roles of the amino acids occupying the S3 pocket were not widely assessed. Herein we report the synthesis and biological evaluation of a set of novel Michael acceptors bearing amino acids of increasing size at the P3 site (1a-g/2a-g, SPR20-SPR33) against FP-2, RD, P. falciparum, and T. brucei. Overall, the Michael acceptors bearing small amino acids at the P3 site exhibited the most potent inhibitory properties towards FP-2. In contrast, analogues with bulky residues at the P3 position were very potent rhodesain inhibitors. In cell based assays, single-digit micromolar EC50 values against the two protozoa were observed. These findings can be a starting point for the development of peptide-based FP-2 and RD inhibitors.

Subnanomolar Cathepsin S Inhibitors with High Selectivity: Optimizing Covalent Reversible α-Fluorovinylsulfones and α-Sulfonates as Potential Immunomodulators in Cancer.

The cysteine protease cathepsin S (CatS) is overexpressed in many tumors. It is known to be involved in tumor progression as well as antigen processing in antigen-presenting cells (APC). Recent evidence suggests that silencing CatS improves the anti-tumor immune response in several cancers. Therefore, CatS is an interesting target to modulate the immune response in these diseases. Here, we present a series of covalent-reversible CatS inhibitors based on the α-fluorovinylsulfone and -sulfonate warheads. We optimized two lead structures by molecular docking approaches, resulting in 22 final compounds which were evaluated in fluorometric enzyme assays for CatS inhibition and for selectivity towards the off-targets CatB and CatL. The most potent inhibitor in the series has subnanomolar affinity (Ki =0.08 nM) and more than 100,000-fold selectivity towards cathepsins B and L. These new reversible and non-cytotoxic inhibitors could serve as interesting leads to develop new immunomodulators in cancer therapy.