Imidazolyl Lipids Enhanced LNP Endosomal Escape for Ferroptosis RNAi Treatment of Cancer.

Treatments for cancer that incorporate small interfering RNA (siRNA) to target iron-dependent ferroptosis are thought to be highly promising. However, creating a reliable and clinically feasible siRNA delivery system continues to be a major obstacle in the field of cancer treatment. Here, three imidazole-based ionizable lipid nanoparticles (LNPs) with pH-sensitive effects are rationally designed and synthesized for siRNA delivery. LNPs formulated with the top-performing lipid (O12-D3-I3) encapsulating FVII siRNA (FVII@O-LNP) elicited greater gene silencing than those with the benchmark Onpattro lipid DLin-MC3-DMA (MC3) due to its stronger endosomal escape. Moreover, Fc-siRNA@O-LNPs encapsulated with ferrocene (Fc) and SLC7A11/Nrf2-targeted siRNA is formulated. The outcomes demonstrate optimal safety profiles and a significant anti-tumor effect by inducing long-lasting and efficient ferroptosis through a synergistic action in vivo. In summary, this work shows that imidazolyl lipid-prepared LNPs are efficient delivery vehicles for cancer therapy and ferroptosis-targeting siRNA administration, both of which have extensive clinical application potential.

Design, Synthesis, and Biological Evaluation of the Quorum-Sensing Inhibitors of Pseudomonas aeruginosa PAO1.

Due to the resistance of Gram-negative bacteria Pseudomonas aeruginosa PAO1 to most clinically relevant antimicrobials, the use of traditional antibiotic treatments in hospitals is challenging. The formation of biofilms, which is regulated by the quorum-sensing (QS) system of Pseudomonas aeruginosa (PA), is an important cause of drug resistance. There are three main QS systems in P. aeruginosa: the las system, the rhl system, and the pqs system. The inhibitors of the las system are the most studied. Previously, the compound AOZ-1 was found to have a certain inhibitory effect on the las system when screened. In this study, twenty-four compounds were designed and synthesized by modifying the Linker and Rings of AOZ-1. Using C. violaceum CV026 as a reporter strain, this study first assessed the inhibitory effects of new compounds against QS, and their SAR was investigated. Then, based on the SAR analysis of compound AOZ-1 derivatives, the parent core of AOZ-1 was replaced to explore the structural diversity. Then, nine new compounds were designed and synthesized with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one. The compound Y-31 (IC50 = 91.55 ± 3.35 µM) was found to inhibit the QS of C. violaceum CV026. Its inhibitory effect on C. violaceum CV026 was better than that of compound AOZ-1 (IC50 > 200 µM). Furthermore, biofilm formation is one of the important causes of Pseudomonas aeruginosa PAO1 resistance. In this study, it was found that compound Y-31, with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one, had the highest biofilm inhibition rate (40.44%). The compound Y-31 has a certain inhibitory effect on the production of PAO1 virulence factors (pyocyanin, rhamnolipid, and elastase) and swarming. When the concentration of compound Y-31 was 162.5 µM, the inhibition rates of pyocyanin, rhamnolipid, and elastase were 22.48%, 6.13%, and 22.67%, respectively. In vivo, the lifetime of wildtype Caenorhabditis elegans N2 infected with P. aeruginosa PAO1 was markedly extended by the new parent nucleus Y-31. This study also performed cytotoxicity experiments and in vivo pharmacokinetics experiments on the compound Y-31. In conclusion, this study identified a compound, Y-31, with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one, which is a potential agent for treating P. aeruginosa PAO1 that is resistant to antibiotics and offers a way to discover novel antibacterial medications.

Discovery of Novel STING Inhibitors Based on the Structure of the Mouse STING Agonist DMXAA.

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.

Design, synthesis and biological activity evaluation of a series of bardoxolone methyl prodrugs.

Bardoxolone methyl (CDDO-Me) has exhibited positive therapeutic effects in clinical trials for diabetic nephropathy (DN), but serious safety risks in the heart exist because of the potential off-target response resulting from the highly active part of CDDO-Me. Herein, we reported a novel strategy to prepare Cathepsin B (CTSB) cleavable and improved water-soluble prodrugs of CDDO-Me. CTSB linkers connection to the highly active α-cyano-α, ß-unsaturated ketone (CUK) part of CDDO-Me with the incorporation of polyethylene glycol (PEG) moieties, provided a series of prodrugs of CDDO-Me without CUK part exposure. Theoretically, these prodrugs shielding CUK part can be stably circulated and finally cleaved by CTSB in lysosomes to release CDDO-Me. In this paper, preliminary curative effects and safety of all prodrugs were determined. Wherein, prodrug 20 displayed relatively better activities than other prodrugs in inhibiting the release of NO from RAW264.7 cells, activating Keap1-Nrf2-ARE signaling pathway and inhibiting NF-κB signaling pathway, which were comparable to CDDO-Me. More importantly, prodrug 20 showed relatively lower human ether-a-go-go-related gene (hERG) inhibitory activity compared with CDDO-Me, which demonstrated prodrug 20 might be safer than CDDO-Me. In conclusion, the novel strategy of shielding CUK part with CTSB linkers provided a new idea for solving the limitations of CDDO-Me in clinical application.

Design, Synthesis, Bioactivity Evaluation, Crystal Structures, and In Silico Studies of New α-Amino Amide Derivatives as Potential Histone Deacetylase 6 Inhibitors.

Hydroxamate, as a zinc-binding group (ZBG), prevails in the design of histone deacetylase 6(HDAC6) inhibitors due to its remarkable zinc-chelating capability. However, hydroxamate-associated genotoxicity and mutagenicity have limited the widespread application of corresponding HDAC6 inhibitors in the treatment of human diseases. To avoid such side effects, researchers are searching for novel ZBGs that may be used for the synthesis of HDAC6 inhibitors. In this study, a series of stereoisomeric compounds were designed and synthesized to discover non-hydroxamate HDAC6 inhibitors using α-amino amide as zinc-ion-chelating groups, along with a pair of enantiomeric isomers with inverted L-shaped vertical structure as cap structures. The anti-proliferative activities were determined against HL-60, Hela, and RPMI 8226 cells, and 7a and its stereoisomer 13a exhibited excellent activities against Hela cells with IC50 = 0.31 µM and IC50 = 5.19 µM, respectively. Interestingly, there is a significant difference between the two stereoisomers. Moreover, an evaluation of cytotoxicity toward human normal liver cells HL-7702 indicated its safety for normal cells. X-ray single crystal diffraction was employed to increase insights into molecule structure and activities. It was found that the carbonyl of the amide bond is on the different side from the amino and pyridine nitrogen atoms. To identify possible protein targets to clarify the mechanism of action and biological activity of 7a, a small-scale virtual screen using reverse docking for HDAC isoforms (1-10) was performed and the results showed that HDAC6 was the best receptor for 7a, suggesting that HDAC6 may be a potential target for 7a. The interaction pattern analysis showed that the α-amino amide moiety of 7a coordinated with the zinc ion of HDAC6 in a bidentate chelate manner, which is similar to the chelation pattern of hydroxamic acid. Finally, the molecular dynamics simulation approaches were used to assess the docked complex's conformational stability. In this work, we identified 7a as a potential HDAC6 inhibitor and provide some references for the discovery of non-hydroxamic acid HDAC6 inhibitors.

Phenoxyaromatic Acid Analogues as Novel Radiotherapy Sensitizers: Design, Synthesis and Biological Evaluation.

Radiotherapy is a vital approach for brain tumor treatment. The standard treatment for glioblastoma (GB) is maximal surgical resection combined with radiotherapy and chemotherapy. However, the non-sensitivity of tumor cells in the hypoxic area of solid tumors to radiotherapy may cause radioresistance. Therefore, radiotherapy sensitizers that increase the oxygen concentration within the tumor are promising for increasing the effectiveness of radiation. Inspired by hemoglobin allosteric oxygen release regulators, a series of novel phenoxyacetic acid analogues were designed and synthesized. A numerical method was applied to determine the activity and safety of newly synthesized compounds. In vitro studies on the evaluation of red blood cells revealed that compounds 19c (∆P50 = 45.50 mmHg) and 19t (∆P50 = 44.38 mmHg) improve the oxygen-releasing property effectively compared to positive control efaproxiral (∆P50 = 36.40 mmHg). Preliminary safety evaluation revealed that 19c exhibited no cytotoxicity towards HEK293 and U87MG cells, while 19t was cytotoxic toward both cells with no selectivity. An in vivo activity assay confirmed that 19c exhibited a radiosensitization effect on orthotopically transplanted GB in mouse brains. Moreover, a pharmacokinetic study in rats showed that 19c was orally available.

Design, synthesis and biological evaluation of acridone analogues as novel STING receptor agonists.

STING (Stimulator of Interferon Genes) has become a focal point in immunology research and a target in drug discovery. The discovery of a potent human-STING agonist is expected to revolutionize current anti-virus or cancer immunotherapy. Inspired by the structure and function of murine STING-specific agonists (DMXAA and CMA), we rationally designed and synthesized four series of novel compounds for the enhancement of human sensitivity. In the cell-based assay, we identified six compounds from all the synthetic small molecules: 2g, 9g, and 12b are STING agonists that are efficacious across species, and all have the skeleton of acridone; 1b, 1c, and 12c just function in the murine STING pathway. Notably, 12b exhibits the best activity among the six agonists, and its inductions of both human and murine STING-dependent signalling are similar to that of 2'3'-cGAMP, which is a well-known STING inducer. While a protein assay indicated that 2 g, 9 g, and 12b could activate the pathway by directly binding human STING, 12b also displayed the strongest binding affinity. Additionally, our studies show that 12b can induce faster, more powerful, and more durable responses of assorted cytokines in a native system than 2'3'-cGAMP. Consequently, our team is the first to successfully modify murine STING agonists to obtain human sensitivity, and these results suggest that 12b is a potent direct-human-STING agonist. Additionally, the acridone analogues demonstrate tremendous potential in the treatment of tumours or viral infections.

Design, Synthesis, and Renal Targeting of Methylprednisolone-Lysozyme.

Methylprednisolone (MP) is often used in the treatment of various kidney diseases, but overcoming the systemic side effects caused by its nonspecific distribution in the body is a challenge. This article reports the design, synthesis, and renal targeting of methylprednisolone-lysozyme (MPS-LZM). This conjugate was obtained by covalently linking MP with the renal targeting carrier LZM through a linker containing an ester bond, which could utilize the renal targeting of LZM to deliver MP to renal proximal tubular epithelial cells and effectively release MP. The reaction conditions for the preparation of the conjugate were mild, and the quality was controllable. The number of drug payloads per LZM was 1.1. Cell-level studies have demonstrated the safety and endocytosis of the conjugate. Further pharmacokinetic experiments confirmed that, compared with that of free MP, the conjugate increased the renal exposure (AUC0-t) of active MP from 17.59 to 242.18 h*ng/mL, and the targeting efficiency improved by approximately 14 times. Tissue and organ imaging further revealed that the conjugate could reach the kidneys quickly, and fluorescence could be detected in the kidneys for up to 12 h. This study preliminarily validates the feasibility of a renal targeting design strategy for MPS-LZM, which is expected to provide a new option for improving kidney-specific distribution of glucocorticoids.

Design, Synthesis and Biological Evaluation of 1-Phenyl-2-(phenylamino) Ethanone Derivatives as Novel MCR-1 Inhibitors.

Polymyxins are considered to be the last-line antibiotics that are used to treat infections caused by multidrug-resistant (MDR) gram-negative bacteria; however, the plasmid-mediated transferable colistin resistance gene (mcr-1) has rendered polymyxins ineffective. Therefore, the protein encoded by mcr-1, MCR-1, could be a target for structure-based design of inhibitors to tackle polymyxins resistance. Here, we identified racemic compound 3 as a potential MCR-1 inhibitor by virtual screening, and 26 compound 3 derivatives were synthesized and evaluated in vitro. In the cell-based assay, compound 6g, 6h, 6i, 6n, 6p, 6q, and 6r displayed more potent activity than compound 3. Notably, 25 µΜ of compound 6p or 6q combined with 2 µg·mL-1 colistin could completely inhibit the growth of BL21(DE3) expressing mcr-1, which exhibited the most potent activity. In the enzymatic assay, we elucidate that 6p and 6q could target the MCR-1 to inhibit the activity of the protein. Additionally, a molecular docking study showed that 6p and 6q could interact with Glu246 and Thr285 via hydrogen bonds and occupy well the cavity of the MCR-1 protein. These results may provide a potential avenue to overcome colistin resistance, and provide some valuable information for further investigation on MCR-1 inhibitors.

Hawaiienols A-D, Highly Oxygenated p-Terphenyls from an Insect-Associated Fungus, Paraconiothyrium hawaiiense.

Four new highly oxygenated p-terphenyls, hawaiienols A-D (1-4), have been isolated from cultures of Paraconiothyrium hawaiiense, a fungus associated with the Septobasidium-infected insect Diaspidiotus sp.; their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2-4 were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation and via electronic circular dichroism calculations, respectively. Compound 1 incorporated the first naturally occurring 4,7-dioxatricyclo[3.2.1.03,6]octane unit in its p-terphenyl skeleton and showed cytotoxicity toward six human tumor cell lines.

A novel hydroxyphenyl hydrazone derivate YCL0426 inhibits cancer cell proliferation through sequestering iron.

Cancer cells have an increased requirement for iron than normal cells, and iron chelators are under active consideration for cancer treatment. The metal-sequestering potential and antiproliferative mechanisms of a novel hydroxyphenyl hydrazone derivate YCL0426 were investigated here. Antiproliferative activity of YCL0426 was detected by MTT assay. The iron-sequestering potential was evaluated by ferrozine-Fe(II) sequestering assay and Fe(II) titration assay. Cell-cycle-arresting profile was checked by flow cytometry and the DNA synthesis status was evaluated by BrdU incorporation assay. SW480 cells stably expressing Rad51-EGFP fusion protein were used to evaluate the DNA damaging potential of the compound. The impact of extra Fe(II) supplement on compound activities was also examined. YCL0426 shows significant antiproliferative activity on 15 cancer cell lines with mean IC50 values of 5.25 µmol/l. YCL0426 displayed concentration-dependent Fe(II) sequestering ability in ferrozine-Fe(II) sequestering assay, and induced upregulation of transferrin receptor 1 and divalent metal transporter 1 expression in HepG2 cells, which are genes responsible for Fe(II) uptake. YCL0426 blocked DNA synthesis in BrdU incorporation assay, and arrested cell cycle at S or G1 phase. Besides, YCL0426 induced Rad51 foci formation and histone H2AX phosphorylation with EC50 values of 1.35 and 2.29 µmol/l, respectively, indicating the emergence of DNA damage. All these cellular responses, and even the growth-inhibiting activity of YCL0426, can be readily reversed by Fe(II) repletion, indicating that iron sequestering is responsible, at least in part, for the antiproliferative activity of YCL0426. YCL0426 is a potent iron chelator that exerts significant antiproliferative activities by inducing G1/S arrest and DNA damage.

Design, synthesis and evaluation of 2,2-dimethyl-1,3-dioxolane derivatives as human rhinovirus 3C protease inhibitors.

The human rhinovirus (HRV) is the most significant cause of the common cold all over the world. The maturation and replication of this virus entirely depend on the activity of a virus-encoded 3C protease. Due to the high conservation among different serotypes and the minimal homology existing between 3C protease and known mammalian enzymes, 3C protease has been regarded as an attractive target for the treatment of HRV infections. In this study, we identified a novel (4R,5R)-N4-(2-((3-methoxyphenyl)amino)ethyl)-2,2-dimethyl-N5-(naphthalen-2-yl)-1,3-dioxolane-4,5-dicarboxamide (7a) to be a HRV 3C protease inhibitor via virtual screening. Further research has been focused on the design, synthesis and in vitro biological evaluation of 7a derivatives. The studies revealed that compound 7d has an IC50 value of 2.50±0.7µM against HRV 3C protease, and it thus could serve as a promising compound for the development of novel anti-rhinoviral medicines.

Design, Synthesis, and Structure-Activity Relationship Study of Epoxysuccinyl-Peptide Derivatives as Cathepsin B Inhibitors.

Cathepsin B is a lysosomal cysteine protease involved in many diseases. The present research demonstrates that derivatives of epoxysuccinyl-peptide are effective and selective cathepsin B inhibitors. We synthesized a series of epoxysuccinyl-peptide derivatives based on the well-known cathepsin B inhibitor E64d. Specifically, we substituted the 2-methylpropane group at the R1 position of E64d with a sulfane, such as ethyl(methyl) sulfane or benzyl(methyl) sulfane. We also designed and synthesized a library of molecules with various substituents at the R2 position of E64d to replace 2-methylbutane. By studying the structure-activity relationships of these newly synthesized molecules as cathepsin B inhibitors, we demonstrated that substituting ethyl(methyl) sulfane for 2-methylbutane (R2) of E64d improves the inhibitory activity and selectivity for cathepsin B inhibition. Our new cathepsin B inhibitors were highly effective and selective.

Chlorotheolides A and B, Spiroketals Generated via Diels-Alder Reactions in the Endophytic Fungus Pestalotiopsis theae.

Chlorotheolides A (1) and B (2), two new spiroketals possessing the unique [4,7]methanochromene and dispiro-trione skeletons, respectively, and their putative biosynthetic precursors, 1-undecen-2,3-dicarboxylic acid (3) and maldoxin (4), were isolated from the solid substrate fermentation of the plant endophytic fungus Pestalotiopsis theae (N635). Their structures were elucidated based on NMR spectroscopic data and electronic circular dichroism calculations. Biogenetically, compounds 1 and 2 could be generated from the co-isolated 3 and 4 via Diels-Alder reactions. Chlorotheolide (2) showed an antiproliferative effect against the human tumor cell line HeLa and induced an autophagic process in the cells.

Design, synthesis and SAR study of novel trisubstituted pyrimidine amide derivatives as CCR4 antagonists.

The design, synthesis and structure-activity relationship studies of some novel trisubstituted pyrimidine amide derivatives prepared as CCR4 antagonists are described. The activities of these compounds were evaluated by the CCR4-MDC chemotaxis inhibition assay. Compound 1, which we have previously reported as a potent antagonist of CCR4, was employed as the positive control. The results indicated that most of the synthesized compounds exhibited some chemotaxis inhibition activity against CCR4. Of these new compounds, compounds 6c, 12a and 12b, with IC50 values of 0.064, 0.077 and 0.069 µM, respectively, showed higher or similar activity compared with compound 1 (IC50 of 0.078 µM). These compounds provide a basis for further structural modifications. The systematic structure-activity relationship of these trisubstituted pyrimidine amide derivatives was discussed based on the obtained experimental data. The results from the SAR study may be useful for identifying more potent CCR4 antagonists.

Simultaneous determination of HD56, a novel prodrug, and its active metabolite in cynomolgus monkey plasma using LC-MS/MS for elucidating its pharmacokinetic profile.

An LC-MS/MS method was developed and validated for the simultaneous determination of the carboxylic acid ester precursor HD56 and the active product HD561 in cynomolgus monkey plasma. Then, the pharmacokinetic characteristics of both compounds following single and multiple i.g. administrations in cynomolgus monkeys were elucidated. In the method, chromatographic separation was achieved with a C18 reversed-phase column and the target quantification was carried out by an electrospray ionization (ESI) source coupled with triple quadrupole mess detector in positive ionization mode with multiple reaction monitoring (MRM) approach. Using the quantification method, the in vitro stability of HD56 in plasma and HD56 pharmacokinetic behavior after i.g. administration in cynomolgus monkey were investigated. It was approved that HD56 did convert into HD561 post-administration. The overall systemic exposure of HD561 post-conversion from HD56 accounted for only about 17% of HD56. After repeated administration at the same dose, there was no significant difference in exposure levels of both HD56 and HD561. However, after multiple dosing, the exposure of HD56 tended to decrease while that of HD561 tended to increase, resulting in a 30% in the exposure ratio. Remarkably, with a carboxylesterase (CES) activity profile akin to humans, the observed in vivo pharmacokinetic profile in cynomolgus monkeys holds promise for predicting HD56/HD561 PK profiles in humans.

Discovery of a Double-Stapled Short Peptide as a Long-Acting HIV-1 Inactivator with Potential for Oral Bioavailability.

Different from most antiretroviral drugs that act as passive defenders to inhibit HIV-1 replication inside the host cell, virus inactivators can attack and inactivate HIV-1 virions without relying on their replication cycle. Herein, we describe the discovery of a hydrocarbon double-stapled helix peptide, termed D26. D26 is based on the HIV-1 gp41 protein lentiviral lytic peptide-3 motif (LLP3) sequence, which can efficiently inhibit HIV-1 infection and inactivate cell-free HIV-1 virions. It was noted that D26 was highly resistant to proteolytic degradation and exhibited a remarkably extended in vivo elimination half-life. Additionally, relative to its linear, nonstapled version, D26 exhibited much higher exposure in sanctuary sites for HIV-1. Amazingly, this lead compound also demonstrated detectable oral absorption. Thus, it can be concluded that D26 is a promising candidate for further development as a long-acting, orally applicable HIV-1 inactivator for the treatment of HIV-1 infection.

Design, synthesis and biological evaluation of N-sulfonyl homoserine lactone derivatives as inhibitors of quorum sensing in Chromobacterium violaceum.

A novel series of N-sulfonyl homoserine lactone derivatives 5a-l has been designed, synthesized and evaluated for quorum sensing inhibitory activities towards violacein production. Of the compounds synthesized, compound 5h was found to possess an excellent level of enantiopurity (99.2% e.e.). The results indicated that compounds bearing an ortho substituent on their phenyl ring exhibited excellent levels of inhibitory activity against violacein production. Compounds 5h and 5k in particular, with IC50 values of 1.64 and 1.66 µM, respectively, were identified as promising lead compounds for further structural modification.

Novel substituted heteroaromatic piperazine and piperidine derivatives as inhibitors of human enterovirus 71 and coxsackievirus a16.

A series of substituted heteroaromatic piperazine and piperidine derivatives were found through virtual screening based on the structure of human enterovirus 71 capsid protein VP1. The preliminary biological evaluation revealed that compounds 8e and 9e have potent activity against EV71 and Coxsackievirus A16 with low cytotoxicity.

Discovery of AI-2 Quorum Sensing Inhibitors Targeting the LsrK/HPr Protein-Protein Interaction Site by Molecular Dynamics Simulation, Virtual Screening, and Bioassay Evaluation.

Quorum sensing (QS) is a cell-to-cell communication mechanism that regulates bacterial pathogenicity, biofilm formation, and antibiotic sensitivity. Among the identified quorum sensing, AI-2 QS exists in both Gram-negative and Gram-positive bacteria and is responsible for interspecies communication. Recent studies have highlighted the connection between the phosphotransferase system (PTS) and AI-2 QS, with this link being associated with protein-protein interaction (PPI) between HPr and LsrK. Here, we first discovered several AI-2 QSIs targeting the LsrK/HPr PPI site through molecular dynamics (MD) simulation, virtual screening, and bioassay evaluation. Of the 62 compounds purchased, eight compounds demonstrated significant inhibition in LsrK-based assays and AI-2 QS interference assays. Surface plasmon resonance (SPR) analysis confirmed that the hit compound 4171-0375 specifically bound to the LsrK-N protein (HPr binding domain, KD = 2.51 × 10-5 M), and therefore the LsrK/HPr PPI site. The structure-activity relationships (SARs) emphasized the importance of hydrophobic interactions with the hydrophobic pocket and hydrogen bonds or salt bridges with key residues of LsrK for LsrK/HPr PPI inhibitors. These new AI-2 QSIs, especially 4171-0375, exhibited novel structures, significant LsrK inhibition, and were suitable for structural modification to search for more effective AI-2 QSIs.