Structure-Guided Discovery of PD-1/PD-L1 Interaction Inhibitors: Peptide Design, Screening, and Optimization via Computation-Aided Phage Display Engineering.

Cancer immunotherapy harnesses the immune system to combat tumors and has emerged as a major cancer treatment modality. The PD-1/PD-L1 immune checkpoint modulates interactions between tumor cells and T cells and has been extensively targeted in cancer immunotherapy. However, the monoclonal antibodies known to target this immune checkpoint have considerable side effects, and novel PD-1/PD-L1 inhibitors are therefore required. Herein, a peptide inhibitor to disrupt PD-1/PD-L1 interactions was designed through structure-driven phage display engineering coupled to computational modification and optimization. BetaPb, a novel peptide library constructed by using the known structure of PD-1/PD-L, was used to develop inhibitors against the immune checkpoint, and specific peptides with high affinity toward PD-1 were screened through enzyme-linked immunosorbent assays, homogeneous time-resolved fluorescence, and biolayer interferometry. A potential inhibitor, B8, was preliminarily screened through biopanning. The binding affinity of B8 toward PD-1 was confirmed through computation-aided optimization. Assessment of B8 variants (B8.1, B8.2, B8.3, B8.4, and B8.5) demonstrated their attenuation of PD-1/PD-L1 interactions. B8.4 exhibited the strongest attenuation efficiency at a half-maximal effective concentration of 0.1 µM and the strongest binding affinity to PD-1 (equilibrium dissociation constant = 0.1 µM). B8.4 outperformed the known PD-1/PD-L1 interaction inhibitor PL120131 in disrupting PD-1/PD-L1 interactions, revealing that B8.4 has remarkable potential for modification to yield an antitumor agent. This study provides valuable information for the future development of peptide-based drugs, therapeutics, and immunotherapies for cancer.

Ser38-His93-Asn91 triad confers resistance of JFH1 HCV NS5A-Y93H variant to NS5A inhibitors.

HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct-acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38-His93-Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum) with codes 1ZH1 and 3FQM/3FQQ. FLAG-NS5A-WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3-5A); however, FLAG-NS5A-Y93H was able to form dimer without the aid of NS3-5A. The Ser38-His93-Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1-NS5A-1ZH1 and -3FQM homology dimers depended on each other for existence and that both NS5A-WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A-Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38-His93-Asn91 triad in resistance of the Y93H variant to NS5A inhibitors.

Xin-yi-san contains potent human CYP1A2 inhibitors and its combined use with theophylline in treatment increases adverse risks in patients.

BACKGROUND: The Xin-yi-san herbal decoction (XYS) is commonly used to treat patients with allergic rhinitis in Taiwan. Theophylline is primarily oxidized with high affinity by human cytochrome P450 (CYP)1A2, and has a narrow therapeutic index. PURPOSE: This study aimed to investigate the inhibition of human CYP1A2-catalyzed theophylline oxidation (THO) by XYS and its adverse effects in patients. METHODS: Human CYPs were studied in recombinant enzyme systems. The influence of concurrent XYS usage in theophylline-treated patients was retrospectively analyzed. RESULTS: Among the major human hepatic and respiratory CYPs, XYS inhibitors preferentially inhibited CYP1A2 activity, which determined the elimination and side effects of theophylline. Among the herbal components of XYS decoction, Angelicae Dahuricae Radix contained potent THO inhibitors. Furanocoumarin imperatorin was abundant in XYS and Angelicae Dahuricae Radix decoctions, and non-competitively inhibited THO activity with Ki values of 77‒84 nM, higher than those (20‒52 nM) of fluvoxamine, which clinically interacted with theophylline. Compared with imperatorin, the intestinal bacterial metabolite xanthotoxol caused weaker THO inhibition. Consistent with the potency of the inhibitory effects, the docking analysis generated Gold fitness values in the order-fluvoxamine > imperatorin > xanthotoxol. During 2017‒2018, 2.6 % of 201,093 theophylline users consumed XYS. After inverse probability weighting, XYS users had a higher occurrence of undesired effects than non-XYS users; in particular, there was an approximately two-fold higher occurrence of headaches (odds ratio (OR), 2.14; 95 % confidence interval (CI), 1.99‒2.30; p < 0.001) and tachycardia (OR, 1.83; 95 % CI, 1.21‒2.77; p < 0.05). The incidence of irregular heartbeats increased (OR, 1.36; 95 % CI, 1.07‒1.72; p < 0.05) only in the theophylline users who took a high cumulative dose (≥ 24 g) of XYS. However, the mortality in theophylline users concurrently taking XYS was lower than that in non-XYS users (OR, 0.24; 95 % CI, 0.14‒0.40; p < 0.001). CONCLUSION: XYS contains human CYP1A2 inhibitors, and undesirable effects were observed in patients receiving both theophylline and XYS. Further human studies are essential to reduce mortality and to adjust the dosage of theophylline in XYS users.

Discovery of oral chemotherapeutic reversal agents for treating multidrug resistance cancer.

The major limitation of cancer treatment is multidrug resistance (MDR), which leads to the inactivation of chemotherapeutic drugs and greater than 90% mortality. To solve this ordeal, we applied ligand-based drug design and bioiosteric replacement strategy from an indazole to a pyrazole ring to discover compounds 27 and 43 with good potential for reversing drug resistance in combination with paclitaxel, and their reversal fold values were 53.2 and 51.0 at 5 µM, respectively, against an MDR cancer cell line (KBvin). Based on the PK profile results, we selected compound 43 with a longer half-life for mechanistic and animal experiments. Combination treatment with compound 43 and paclitaxel-induced apoptosis and enhanced subG1 by decreasing mitochondrial membrane potential in KBvin cells. In addition, 43 also inhibited P-gp function by interfering with ATPase activity. Meanwhile, cotreatment with compound 43 and paclitaxel significantly suppressed tumor growth (TGI = 55.5%) at a dose of 200 mg/kg (PO) in a xenograft model and showed no obvious liver or kidney toxicity by H&E staining. Overall, compound 43 may serve as a safe and effective oral resistance reversal chemotherapeutic agent.

Structure-guided identification and characterization of potent inhibitors targeting PhoP and MtrA to combat mycobacteria.

Mycobacteria are causative agents of tuberculosis (TB), which is a global health concern. Drug-resistant TB strains are rapidly emerging, thereby necessitating the urgent development of new drugs. Two-component signal transduction systems (TCSs) are signaling pathways involved in the regulation of various bacterial behaviors and responses to environmental stimuli. Applying specific inhibitors of TCSs can disrupt bacterial signaling, growth, and virulence, and can help combat drug-resistant TB. We conducted a comprehensive pharmacophore-based inhibitor screening and biochemical and biophysical examinations to identify, characterize, and validate potential inhibitors targeting the response regulators PhoP and MtrA of mycobacteria. The constructed pharmacophore model Phar-PR-n4 identified effective inhibitors of formation of the PhoP-DNA complex: ST132 (IC50 = 29 ± 1.6 µM) and ST166 (IC50 = 18 ± 1.3 µM). ST166 (KD = 18.4 ± 4.3 µM) and ST132 (KD = 14.5 ± 0.1 µM) strongly targeted PhoP in a slow-on, slow-off manner. The inhibitory potency and binding affinity of ST166 and ST132 for MtrAC were comparable to those of PhoP. Structural analyses and molecular dynamics simulations revealed that ST166 and ST132 mainly interact with the α8-helix and C-terminal ß-hairpin of PhoP, with functionally essential residue hotspots for structure-based inhibitor optimization. Moreover, ST166 has in vitro antibacterial activity against Macrobacterium marinum. Thus, ST166, with its characteristic 1,2,5,6-tetrathiocane and terminal sulphonic groups, has excellent potential as a candidate for the development of novel antimicrobial agents to combat pathogenic mycobacteria.