Antibody-drug conjugate (ADC) is a novel efficient drug delivery system that has been successfully used in clinical practice, and it has become a research hotspot in the anti-tumor drug field. Acid-cleavable linkers were first used in clinical ADCs, but their structural variety (e.g., hydrazone and carbonate) is still limited, and their stability is usually insufficient. Designing novel acid-cleavable linkers for the conjugation of the popular cytotoxin monomethyl auristatin E (MMAE) has always been a significant topic. In this paper, we generate a novel, silyl ether-based acid-cleavable antibody-MMAE conjugate, which skillfully achieves efficient combination of amino-conjugated MMAE with the acid-triggered silyl ether group by introducing p-hydroxybenzyl alcohol (PHB). The stability, acid-dependence cleavage, effective mechanism, efficacy and safety of the resulting ADC were systematically studied; the results show that it exhibits a significant improvement in stability, while maintaining appropriate efficacy and controlled therapeutic toxicity. This strategy is expected to expand a new type of acid-cleavable linkers for the development of ADCs with highly potent payloads.

Monomethyl auristatin E (MMAE) is the most popular and widely used cytotoxin in the development of antibody-drug conjugates (ADCs). However, current MMAE-based ADCs are all constructed using cleavable linkers, and this design concept still has insurmountable drawbacks. Their potential instabilities and lipophilic MMAE-induced “bystander effect” inevitably increase the toxicity to normal tissues. Herein, we overturn previous negative views of MMAE-based ADCs with non-cleavable linkers and propose using ionized L-Cysteine (Cys)-linker-MMAE as a novel payload, which can ingeniously enrich and enter tumor cells through receptor-mediated endocytosis of antibodies while its lower permeability helps to avoid further off-target toxicity. We demonstrate that Cys-linker-MMAE maintains high potency similar to free MMAE at the tubulin molecular level and can also be efficiently released in target cells. As a result, the preferred ADC (mil40-15) not only exhibits ideal plasma stability and maintains potent cytotoxicity as MMAE (IC₅₀: 10⁻¹¹ M), but also shows improved safety with lower bystander toxicity (IC₅₀: 10⁻⁹ M), its maximum tolerated dose approaching the level of the naked antibody (160 mg/kg). This study indicated that Cys-linker-MMAE has the potential as a potent payload for ADCs, which is expected to provide novel strategies for the development of MMAE-based ADCs.

Enterovirus infections can cause hand, foot, and mouth disease (HFDM), aseptic meningitis, encephalitis, myocarditis, and acute flaccid myelitis, leading to death of infants and young children. However, no specific antiviral drug is currently available for the treatment of this type of infection. The Unites States and United Kingdom health authorities recently approved a new antiviral drug, molnupiravir, for the treatment of COVID-19. In this study, we reported that molnupiravir (EIDD-2801) and its active form, EIDD-1931, have broad-spectrum anti-enterovirus potential. Our data showed that EIDD-1931 could significantly reduce the production of EV-A71 progeny virus and the expression of EV-A71 viral protein at non-cytotoxic concentrations. The results of the time-of-addition assay suggest that EIDD-1931 acts at the post-entry step, which is in accordance with its antiviral mechanism. The intraperitoneal administration of EIDD-1931 and EIDD-2801 protected 1-day-old ICR suckling mice from lethal EV-A71 challenge by reducing the viral load in various tissues of the infected mice. The pharmacokinetics analysis indicated that the plasma drug concentration overwhelmed the EC₅₀ for enteroviruses, suggesting the clinical potential of molnupiravir against enteroviruses. Thus, molnupiravir along with its active form, EIDD-1931, may be a promising drug candidate against enterovirus infections.

The stimulator-of-interferon-gene (STING) protein is involved in innate immunity. The drug DMXAA (5,6-dimethylxanthenone-4-acetic acid) proved to be a potent murine-STING (mSTING) agonist but had little effect on human-STING (hSTING). In this paper, we draw upon the comparison of different crystal structures and protein-ligand interaction relationships analysis to venture the hypothesis that the drug design of DMXAA variants has the potential to convert STING agonists to inhibitors. Based on our previous discovery of two DMXAA analogs, 3 and 4 (both could bind to STING), we structurally optimized them and synthesized new derivatives, respectively. In binding assays, we found compounds 11 and 27 to represent STING binders that were superior to the original structures and discussed the structure-activity relationships. All target compounds were inactive in cellular assays for the screening of STING agonistic activity. Gratifyingly, we identified 11 and 27 as STING inhibitors with micromolar activity in both hSTING and mSTING pathways. In addition, 11 and 27 inhibited the induction of interferon and inflammatory cytokines activated by 2′3′-cGAMP without apparent cytotoxicity. These findings break the rigid thinking that DMXAA provides the structural basis specifically for STING agonists and open up more possibilities for developing novel STING agonists or inhibitors.

The PB2 subunit of the influenza RNA-dependent RNA polymerase (RdRp) has been identified as a promising target for the treatment of influenza. To expand the chemical space of the known influenza polymerase PB2 inhibitor–pimodivir (formerly VX-787) and improve its pharmacokinetic profile, two pimodivir analogs containing 2,3-dihydro-imidazopyridine fragment (comp. I and comp. II) were designed, synthesized, and evaluated for anti-influenza virus activity. In the cytopathic effect (CPE) inhibition assay, comp. I and comp. II showed IC₅₀ values of 0.07 and 0.09 μM for A/Puerto Rico/8/34 (H1N1) and 0.04 and 0.07 μM for A/Hong Kong/8/68 (H3N2), respectively. Protein-binding affinity assay results showed a concentration-dependent association and dissociation pattern, with K_D values of 1.398 and 1.670 μM, respectively. In vitro metabolic stability assays showed that comp. I and comp. II exhibited good stability to liver microsomes and considerably less sensitivity to aldehyde oxidase compared to pimodivir. The binding modes of comp. I and comp. II were similar to those of VX-787; however, comp. I and comp. II had lower structural adaptability to PB2 than VX-787. Our results provide helpful information regarding the structure–activity relationship for the design of novel PB2 inhibitors and a reference for the development of drugs containing 2,3-dihydro-imidazopyridine fragments.

Dengue virus (DENV) is a major mosquito-borne human pathogen in tropical countries; however, there are currently no targeted antiviral treatments for DENV infection. Compounds 27 and 29 have been reported to be allosteric inhibitors of DENV RdRp with potent inhibitory effects. In this study, the structures of compounds 27 and 29 were optimized using computer-aided drug design (CADD) approaches. Nine novel compounds were synthesized based on rational considerations, including molecular docking scores, free energy of binding to receptor proteins, predicted Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) parameters, structural diversity, and feasibility of synthesis. Subsequently, the anti-DENV activity was assessed. In the cytopathic effect (CPE) assay conducted on BHK-21 cells using the DENV2 NGC strain, both SW-b and SW-d demonstrated comparable or superior activity against DENV2, with IC₅₀ values of 3.58 ± 0.29 μM and 23.94 ± 1.00 μM, respectively, compared to that of compound 27 (IC₅₀ = 19.67 ± 1.12 μM). Importantly, both SW-b and SW-d exhibited low cytotoxicity, with CC₅₀ values of 24.65 μmol and 133.70 μmol, respectively, resulting in selectivity indices of 6.89 and 5.58, respectively. Furthermore, when compared to the positive control compound 3′-dATP (IC₅₀ = 30.09 ± 8.26 μM), SW-b and SW-d displayed superior inhibitory activity in an enzyme inhibitory assay, with IC₅₀ values of 11.54 ± 1.30 μM and 13.54 ± 0.32 μM, respectively. Molecular dynamics (MD) simulations elucidated the mode of action of SW-b and SW-d, highlighting their ability to enhance π–π packing interactions between benzene rings and residue W795 in the S1 fragment, compared to compounds 27 and 29. Although the transacylsulphonamide fragment reduced the interaction between T794 and NH, it augmented the interaction between R729 and T794. In summary, our study underscores the potential of SW-b and SW-d as allosteric inhibitors targeting the DENV NS5 RdRp domain. However, further in vivo studies are warranted to assess their pharmacology and toxicity profiles.