Search

Herein, we describe the X-ray structure-based designand optimization of biaryl mannoside FimH inhibitors. Diverse modificationsto the biaryl ring to improve druglike physical and pharmacokineticproperties of mannosides were assessed for FimH binding affinity basedon their effects on hemagglutination and biofilm formation along withdirect FimH binding assays. Substitution on the mannoside phenyl ringortho to the glycosidic bond results in large potency enhancementsseveral-fold higher than those of corresponding unsubstituted matchedpairs and can be rationalized from increased hydrophobic interactionswith the FimH hydrophobic ridge (Ile13) or “tyrosine gate”(Tyr137 and Tyr48) also lined by Ile52. The lead mannosides have increasedmetabolic stability and oral bioavailability as determined from invitro PAMPA predictive model of cellularpermeability and in vivo pharmacokinetic studies in mice, therebyrepresenting advanced preclinical candidates with promising potentialas novel therapeutics for the clinical treatment and prevention ofrecurring urinary tract infections. [ABSTRACT FROM AUTHOR]

The stress kinase MAPK13 (aka p38δ-MAPK) is an attractive entry point for therapeutic intervention because it regulates the structural remodeling that can develop after epithelial barrier injury in the lung and likely other tissue sites. However, a selective, safe, and effective MAPK13 inhibitor is not yet available for experimental or clinical application. Here we identify a first-in-kind MAPK13 inhibitor using structure-based drug design combined with a screening funnel for cell safety and molecular specificity. This inhibitor (designated NuP-4) down-regulates basal-epithelial stem cell reprogramming, structural remodeling, and pathophysiology equivalently to Mapk13 gene-knockout in mouse and mouse organoid models of post-viral lung disease. This therapeutic benefit persists after stopping treatment as a sign of disease modification and attenuates key aspects of inflammation and remodeling as an indication of disease reversal. Similarly, NuP-4 treatment can directly control cytokine-stimulated growth, immune activation, and mucinous differentiation in human basal-cell organoids. The data thereby provide a new tool and potential fix for long-term stem cell reprogramming after viral injury and related conditions that require MAPK13 induction-activation.

Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively downregulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease. NEW & NOTEWORTHY This study describes the discovery of a potent mitogen-activated protein kinase 13-14 (MAPK13-14) inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases [e.g., asthma, chronic obstructive pulmonary disease (COPD), Covid-19, postviral, and allergic respiratory disease] and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.

Targeted therapeutic agents, such as inhibitors of epithelial growth factor receptor (EGFR), have transformed the management of non-small cell lung cancer (NSCLC) patients. MET-amplified NSCLC cells display resistance to EGFR-targeting agents, but are addicted to MET signaling for survival and proliferation and are sensitive to MET inhibition. However, responsive cancer cells invariably develop resistance to MET-targeted treatment. The tumor microenvironment plays a major role in resistance to anticancer therapy. We demonstrated that fibroblasts block the response of MET-amplified NSCLC cells to the MET kinase inhibitor, JNJ38877605 in an HGF-dependent manner. Thus, MET-amplified NSCLC cells become addicted to HGF upon pharmacological inhibition of MET. HGF restored phosphorylation of MET, EGFR and RON, and maintained pro-survival AKT and ERK signaling in MET-inhibited cells. We developed a small molecule inhibitor of pro-HGF activation, SRI31215, which acts as a triplex inhibitor of the pro-HGF activating proteases matriptase, hepsin and HGF activator (HGFA). SRI31215 blocked crosstalk between tumor cells and fibroblasts and overcame fibroblast-mediated resistance to MET inhibition by preventing fibroblast-mediated reactivation of AKT and ERK signaling. Structurally unrelated triplex inhibitors of matriptase, hepsin and HGFA that we developed in parallel showed similar biological activity. Our data suggest that simultaneous inhibition of HGF and MET is required to overcome resistance to MET inhibitors in MET-amplified NSCLC cells. This provides a rationale for the development of novel combination therapeutic strategies for the treatment of NSCLC patients with MET amplification., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the O-glycosidic bond (O-mannosides). Herein, we disclose that compounds having the O-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked C-mannosides that show improved pharmacokinetic (PK) properties relative to O-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the R-hydroxy isomer has a 60-fold increase in potency. This new class of C-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI.

Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON.

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial urinary tract infections (UTIs) and pose significant clinical challenges. These infections are polymicrobial in nature and are often associated with multidrug-resistant pathogens, including uropathogenic Escherichia coli (UPEC). Urinary catheterization elicits major histological and immunological alterations in the bladder that can favor microbial colonization and dissemination in the urinary tract. We report that these biological perturbations impact UPEC pathogenesis and that bacterial reservoirs established during a previous UPEC infection, in which bacteriuria had resolved, can serve as a nidus for subsequent urinary catheter colonization. Mannosides, small molecule inhibitors of the type 1 pilus adhesin, FimH, provided significant protection against UPEC CAUTI by preventing bacterial invasion and shifting the UPEC niche primarily to the extracellular milieu and on the foreign body. By doing so, mannosides potentiated the action of trimethoprim-sulfamethoxazole in the prevention and treatment of CAUTI. In this study, we provide novel insights into UPEC pathogenesis in the context of urinary catheterization, and demonstrate the efficacy of novel therapies that target critical mechanisms for this infection. Thus, we establish a proof-of-principle for the development of mannosides to prevent and eventually treat these infections in the face of rising antibiotic-resistant uropathogens.

FimH-mediated cellular adhesion to mannosylated proteins is critical in the ability of uropathogenic E. coli (UPEC) to colonize and invade the bladder epithelium during urinary tract infection. We describe the discovery and optimization of potent small-molecule FimH bacterial adhesion antagonists based on alpha-d-mannose 1-position anomeric glycosides using X-ray structure-guided drug design. Optimized biarylmannosides display low nanomolar binding affinity for FimH in a fluorescence polarization assay and submicromolar cellular activity in a hemagglutination (HA) functional cell assay of bacterial adhesion. X-ray crystallography demonstrates that the biphenyl moiety makes several key interactions with the outer surface of FimH including pi-pi interactions with Tyr-48 and an H-bonding electrostatic interaction with the Arg-98/Glu-50 salt bridge. Dimeric analogues linked through the biaryl ring show an impressive 8-fold increase in potency relative to monomeric matched pairs and represent the most potent FimH antagonists identified to date. The FimH antagonists described herein hold great potential for development as novel therapeutics for the effective treatment of urinary tract infections.

The cobalt-catalyzed cyclotrimerization of bis(4-pyridyl)acetylene affords hexakis(4-pyridyl)benzene in moderate yield. Alkylation with n-butyltriflate gives hexakis(4-(N-butylpyridylium))benzene triflate (1 6+), which can be reduced with Na/Hg in DMF to neutral 10. A single-crystal X-ray diffraction structure reveals that 1 0 has a chair-cyclohexane-like core and a [6]radialene structure. Cyclic voltammetry shows that 1 6+ is reversibly reduced to 1 2+ in one four-electron step and 1 2+ is reversibly reduced to 1 0 in one two-electron step. A reduction by four electrons at one potential is unprecedented for a molecule in which the electrochemically active centers are in electronic communication. The large structural transformation from 1 6+ to 1 0 is responsible for the "potential inversion" in the cyclic voltammetry, and DFT calculations suggest a possible structure for the stable intermediate 1 2+. A comparison is made to the electrochemistry and structural transformations in a previously prepared [4]radialene analogue of 1 0.

[reaction: see text] Catalytic activity of nonaromatic bicyclic amidines and bicyclic isothioureas in acylation reactions was found to be remarkably dependent on the sizes of both rings. DBN and especially its thia-analogue (THTP) have been identified as highly active acylation catalysts.

Treatment of alpha, beta-unsaturated carbonyl compounds or methyl propargyl ether with acylchlorobis(cyclopentadienyl)titanium in the presence of triethylamine and a copper salt in aqueous THF resulted in acylation of the carbon-carbon multiple bond, yielding the corresponding 1,4-diketones in good yields.