Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.
- MeSH
- kathepsin B * antagonisté a inhibitory metabolismus MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- Schistosoma mansoni * enzymologie účinky léků MeSH
- schistosomicidy farmakologie chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Aldo-keto reductase 1C3 (AKR1C3) catalyzes the reduction of androstenedione to testosterone and reduces the effectiveness of chemotherapeutics. AKR1C3 is a target for treatment of breast and prostate cancer and AKR1C3 inhibition could be an effective adjuvant therapy in the context of leukemia and other cancers. In the present study, steroidal bile acid fused tetrazoles were screened for their ability to inhibit AKR1C3. Four C24 bile acids with C-ring fused tetrazoles were moderate to strong AKR1C3 inhibitors (37-88% inhibition), while B-ring fused tetrazoles had no effect on AKR1C3 activity. Based on a fluorescence assay in yeast cells, these four compounds displayed no affinity for estrogen receptor-α, or the androgen receptor, suggesting a lack of estrogenic or androgenic effects. A top inhibitor showed specificity for AKR1C3 over AKR1C2, and inhibited AKR1C3 with an IC50 of ∼7 μM. The structure of AKR1C3·NADP+ in complex with this C-ring fused bile acid tetrazole was determined by X-ray crystallography at 1.4 Å resolution, revealing that the C24 carboxylate is anchored to the catalytic oxyanion site (H117, Y55); meanwhile the tetrazole interacts with a tryptophan (W227) important for steroid recognition. Molecular docking predicts that all four top AKR1C3 inhibitors bind with nearly identical geometry, suggesting that C-ring bile acid fused tetrazoles represent a new class of AKR1C3 inhibitors.
- Publikační typ
- časopisecké články MeSH
Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC50 values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 μM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.
- MeSH
- inhibitory enzymů * chemie MeSH
- krystalografie MeSH
- lidé MeSH
- purinnukleosidfosforylasa * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Influenza virus causes severe respiratory infection in humans. Current antivirotics target three key proteins in the viral life cycle: neuraminidase, the M2 channel and the endonuclease domain of RNA-dependent-RNA polymerase. Due to the development of novel pandemic strains, additional antiviral drugs targetting different viral proteins are still needed. The protein-protein interaction between polymerase subunits PA and PB1 is one such possible target. We recently identified a modified decapeptide derived from the N-terminus of the PB1 subunit with high affinity for the C-terminal part of the PA subunit. Here, we optimized its amino acid hotspots to maintain the inhibitory potency and greatly increase peptide solubility. This allowed thermodynamic characterization of peptide binding to PA. Solving the X-ray structure of the peptide-PA complex provided structural insights into the interaction. Additionally, we optimized intracellular delivery of the peptide using a bicyclic strategy that led to improved inhibition in cell-based assays.
Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
- MeSH
- cytokiny MeSH
- interferony MeSH
- lidé MeSH
- ligandy MeSH
- membránové proteiny * metabolismus MeSH
- myši MeSH
- nukleotidy cyklické * chemie MeSH
- nukleotidyltransferasy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.
- MeSH
- hydraziny chemie farmakologie MeSH
- inhibitory proteas chemie farmakologie MeSH
- kathepsin K antagonisté a inhibitory metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- nádorové buňky kultivované MeSH
- nitrily chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.
Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory metabolismus MeSH
- Candida parapsilosis enzymologie MeSH
- fungální proteiny antagonisté a inhibitory metabolismus MeSH
- inhibitory proteas chemická syntéza chemie farmakologie MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- peptidomimetika chemická syntéza chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Peptidomimetic inhibitors of fibroblast activation protein (FAP) are regarded as promising tools for tumor targeting in vivo. Even though several peptidomimetic compounds with nanomolar potency have been described, broad chemical space for further modification remained unexplored. Therefore, we set to analyze the structure-activity relationship (SAR) of pseudopeptide compound series with α-ketoamide warheads in order to explore the contributions of the P1' and P2' moieties to the inhibitory potency. A series of novel inhibitors bearing varied P1' and/or P2' moieties was synthesized by combining a Passerini reaction-Amine Deprotection-Acyl Migration (PADAM) approach with peptide coupling and subsequent oxidation. The resulting compounds inhibited FAP and the related prolyl endopeptidase (PREP) with potencies in the nanomolar to sub-nanomolar range. The most potent FAP inhibitor IOCB22-AP446 (6d, IC50 = 89 pM) had about 36-fold higher inhibition potency than the most potent inhibitor published to date. The compounds were selective over FAP's closest homolog DPP-IV, were stable in human and mouse plasma and in mouse microsomes, and displayed minimal cytotoxicity in tissue cultures.
- MeSH
- fibroblasty metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- myši MeSH
- prolyloligopeptidasy metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions located in the enzyme's catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3',4'-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC50 values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.
