Potent Synergistic Activity Research Articles

Potent Anti-Influenza Synergistic Activity of Theobromine and Arainosine.

Influenza represents one of the biggest health threats facing humanity. Seasonal epidemics can transition to global pandemics, with cross-species infection presenting a continuous challenge. Although vaccines and several anti-viral options are available, constant genetic drifts and shifts vitiate any of the aforementioned prevention and treatment options. Therefore, we describe an approach targeted at the virus's channel to derive new anti-viral options. Specifically, Influenza A's M2 protein is a well-characterized channel targeted for a long time by aminoadamantane blockers. However, widespread mutations in the protein render the drugs ineffective. Consequently, we started by screening a repurposed drug library against aminoadamantane-sensitive and resistant M2 channels using bacteria-based genetic assays. Subsequent in cellulo testing and structure-activity relationship studies yielded a combination of Theobromine and Arainosine, which exhibits stark anti-viral activity by inhibiting the virus's channel. The drug duo was potent against H1N1 pandemic swine flu, H5N1 pandemic avian flu, aminoadamantane-resistant and sensitive strains alike, exhibiting activity that surpassed Oseltamivir, the leading anti-flu drug on the market. When this drug duo was tested in an animal model, it once more outperformed Oseltamivir, considerably reducing disease symptoms and viral RNA progeny. In conclusion, the outcome of this study represents a new potential treatment option for influenza alongside an approach that is sufficiently general and readily applicable to other viral targets.

Abstract LB126: Generation of a novel ROR1 x CD3 bispecific T-cell engager for better tumor killing and minimal cytokine release

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) expression is a highly specific tumor associated antigen expressed in multiple hematological malignancies and solid tumors. EMB-07 is a novel monovalent asymmetrical tandem Fab (MAT-Fab) antibody composed of two different Fab fragments targeting ROR1 and CD3ε respectively, and a “knob-in-hole” Fc fragment maintaining FcRn binding and Ig-like pharmacokinetics in vivo. To avoid light chain miss-pairing, the anti-ROR1 light chain was directly connected to the N-terminus of the anti-CD3 heavy chain. The ROR1 binding arm has a high affinity of 0.52 nM and recognizes a membrane distal epitope, which is different from a reference BiTE molecule currently in clinical trial. In comparison with this BiTE molecule, EMB-07 showed similar tumor killing efficacy to ROR1 expressing tumor cell lines while it induced much lower levels of cytokines both in vitro and in vivo. Moreover, comparing with a clinical stage ROR1-vc-MMAE ADC, EMB-07 demonstrated ~500-fold increased tumor lysis potency in vitro. In PD-1 resistant tumor xenograft mice models, EMB-07 showed potent tumor growth inhibition and synergistic activity in combination with PD-1 blockade. In a preclinical GLP toxicological study, EMB-07 was well tolerated in cynomolgus monkeys with no evidence of lethality, life-threatening toxicity, or obvious EMB-07-related macroscopic and histopathological adverse findings up to 50 mg/kg. By using a highly specific IHC clone, we validated membrane-associated ROR1 expression in multiple tumor types, including triple negative breast cancer, ovarian cancer, endometrial cancer, lung adenocarcinoma, and pancreatic cancer, while the observation of its expression in normal tissues is very limited. This study supports the clinical development of EMB-07 for treating multiple solid tumors with ROR1 expression. A FIH study of EMB-07 in locally advanced/metastatic solid tumors or relapse/refractory lymphoma is currently ongoing (NCT05607498). Citation Format: Danqing Wu, Shiyong Gong, Xuan Wu, Gaowa Naren, Liqin Dong, Stephan Lensky, Chengbin Wu. Generation of a novel ROR1 x CD3 bispecific T-cell engager for better tumor killing and minimal cytokine release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB126.

Abstract 7168: PBI-410 (GQ1010), A novel Trop-2-targeted ADC demonstrates a favorable safety and toxicokinetic profile in multiple preclinical assessments

Abstract Introduction: Trop-2-targeting ADCs (Trop-2 ADC) have demonstrated notable clinical efficacy; however, safety concerns necessitate frequent dose adjustments or discontinuations. PBI-410 (GQ1010) is a next-generation TROP-2 ADC developed using GeneQuantum's Ligase-Dependent Conjugation (iLDC) technology, that integrates a highly stable linker and a novel camptothecin analogue payload, TopoIx. This study included comprehensive preclinical evaluations that demonstrated a favorable safety and TK profile and potential for broader therapeutic margin versus other Trop-2 ADCs approved or in development. Methods: TopoIx underwent extensive Good Laboratory Practice toxicity assessments in Sprague-Dawley (SD) rats (payload alone) and as part of the intact ADC in Non-Human Primates. Rats were administered repeated intravenous infusions of 0.5, 1.25, and 2.5 mg/kg of H0011 once a week over 5 doses. The potential toxicity profile of PBI-410 was evaluated in cynomolgus monkeys via repeated intravenous infusions of 10, 30, and 60 mg/kg doses every 3 weeks for 3 doses, followed by a six-week recovery phase. Results: TopoIx administration in SD rats exhibited good tolerance, with slight reductions in body weight gain and food consumption. Microscopic tissue changes resolved post-drug withdrawal, except for persistent findings in the sciatic nerve. Repeated doses of PBI-410 in cynomolgus monkeys were well-tolerated, primarily manifesting as skin pigmentation and associated microscopic alterations in specific tissues, such as eyes, esophagus, thymus, and lymph nodes typical for topoisomerase inhibitors. Importantly, these changes were reversible upon drug cessation. The HNSTD/NOAEL for PBI-410 was determined to be 60 mg/kg. This corresponds to mean Cmax of 1760 µg/mL and AUCinf of 235000 µg*hr/mL for ADC and for TopoIx mean Cmax of 16.4 ng/mL and AUCinf of 2610 ng*hr/mL. The lower exposure of TopoIx suggests minimum payload shedding in the systemic circulation. Notably, PBI-410 demonstrated a significantly enhanced safety profile compared to other Topo1-based Trop-2 ADCs, including an absence of hematopoietic and intestinal toxicities, and no pulmonary toxicity or interstitial lung disease (ILD) observed. Conclusion: PBI-410 demonstrated a favorable and differentiated nonclinical safety profile and favorable TK relative to those reported to date for other Trop-2 ADCs in development. This profile, in addition to the potent anti-tumor response and synergistic activity in combination with anti-PD1 reported previously, position PBI-410 as a best-in-class potential Trop-2 ADC and partner of choice for anti-PD1 combination therapy. Additionally, TopoIx has displayed excellent properties among the TOP1 inhibitors, making it suitable for a broader therapeutic application based on exceptional potency while maintaining a favorable safety profile. Citation Format: Yajun Sun, Vijayapal Reddy, Palaniappan Kulanthaivel, Yanwen Feng, Jie Zhao, Zhiyan Chi, Junhao Wang, Lili Shi, Gang Qin, Paul H. Song. PBI-410 (GQ1010), A novel Trop-2-targeted ADC demonstrates a favorable safety and toxicokinetic profile in multiple preclinical assessments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7168.

Synergistic effects of thyme and oregano essential oil combinations for enhanced functional properties of sericin/pectin film

This work aimed to assess the synergistic antibacterial effects of thyme and oregano essential oils in various ratios (thyme:oregano; 10:0, 8:2, 6:4, 4:6, 2:8, 10:0). We hypothesized that the synergistic combination of thyme and oregano essential oils can be effectively incorporated into sericin/pectin film to enhance its functional properties. Among the combinations tested, the mixture of thyme/oregano essential oil (TOE) at an 8:2 ratio exhibited the most potent synergistic activity against P. aeruginosa and S. aureus, with fractional inhibitory concentration index (FICindex) of 0.9. In this combination, thymol constituting 51.83 % of TOE (8:2), was the predominant component. TOE at an 8:2 ratio was selected to incorporate into sericin/pectin film. Different concentrations of TOE (0.8 %, 1.2 % and 1.6 %) were applied to evaluate their impact on film properties compared to a film without essential oil (control). It was found that increasing TOE concentration (control; 0 %) to 1.6 % reduced film moisture content (from 21.53 % to 16.91 %), decreased yellowness (from 18.24 to 15.92), diminished gloss (from 63.79 to 11.18), lowered swelling index (from 1.24 to 0.98), and reduced tensile strength (from 9.70 to 4.14 MPa). However, the addition of TOE showed higher film total phenolic content (8.59–31.53 mg gallic acid/g dry sample) and increased antioxidant activity (0.99–3.68 μmol Trolox /g dry sample). Moreover, the film with 1.2 % and 1.6 % of thyme/oregano essential oil exhibited inhibitory effects against all tested bacteria. Therefore, the thyme/oregano essential oil combination can provide the desirable physicochemical properties of the sericin/pectin film, as well as its antibacterial and antioxidant activities, making it a promising alternative for food packaging material applications.

Combining SNAPs with antibiotics shows enhanced synergistic efficacy against S. aureus and P. aeruginosa biofilms

Biofilm infections are associated with a high mortality risk for patients. Antibiotics perform poorly against biofilm communities, so high doses and prolonged treatments are often used in clinical settings. We investigated the pairwise interactions of two synthetic nano-engineered antimicrobial polymers (SNAPs). The g-D50 copolymer was synergistic with penicillin and silver sulfadiazine against planktonic Staphylococcus aureus USA300 in synthetic wound fluid. Furthermore, the combination of g-D50 and silver sulfadiazine showed a potent synergistic antibiofilm activity against S. aureus USA300 using in vitro and ex vivo wound biofilm models. The a-T50 copolymer was synergistic with colistin against planktonic Pseudomonas aeruginosa in synthetic cystic fibrosis medium, and this pair showed a potent synergistic antibiofilm activity against P. aeruginosa in an ex vivo cystic fibrosis lung model. SNAPs thus have the potential for increased antibiofilm performance in combination with certain antibiotics to shorten prolonged treatments and reduce dosages against biofilm infection.

A phase 3, two-stage, randomized study of mezigdomide, carfilzomib, and dexamethasone (MeziKd) versus carfilzomib and dexamethasone (Kd) in relapsed/refractory multiple myeloma (RRMM): SUCCESSOR-2.

TPS8070 Background: Mezigdomide (MEZI), a novel oral cereblon E3 ligase modulator (CELMoD), induces maximal degradation of Ikaros/Aiolos leading to increased MM cell apoptosis and immune-stimulatory effects. MeziKd has shown potent synergistic antiproliferative activity in MM cell lines resistant to lenalidomide (LEN), and in a phase 1/2 study showed promising preliminary efficacy and safety in RRMM. The SUCCESSOR-2 phase 3 trial (NCT05552976) will compare the efficacy and safety of MeziKd vs Kd in patients (pts) with RRMM. Methods: This multicenter, open-label study comprises 2 stages. In Stage 1, ≥ 128 pts will be randomized 3:3:3:2 to 1 of 3 MEZI doses (1.0, 0.6, or 0.3 mg) + Kd, or to the Kd arm to select the optimal MEZI dose in combination with Kd for Stage 2. In Stage 2, ≈ 397 additional pts will be randomized 3:2 to MeziKd at the selected MEZI dose or to Kd, for efficacy and safety analyses (Stage 1 pts in the selected MeziKd dose cohort and Kd arm will also be included in these analyses). Pts will be stratified by age (≤ 70 vs > 70 y), number of prior lines of treatment (Tx; ≤ 2 vs > 2), and ISS stage at screening (I vs II vs III). MEZI dose selection will be based on Stage 1 efficacy, safety, pharmacokinetic and pharmacodynamic data, and exposure–response analyses. Primary efficacy endpoint is progression-free survival (PFS). Assuming a decrease in PFS risk by 33.3% (HR = 0.667) with MeziKd, under exponential distribution assumption of PFS (1-sided α = 0.025) and adjusted for 3 interim analyses, ≥ 273 PFS events will have ≈ 89% power to detect improvement in Tx effect. The planned interim analyses are for: MEZI dose selection at end of Stage 1, and to examine PFS futility and superiority when ≈ 82 (30%) and ≈ 205 (75%) events, respectively, have been accumulated. Secondary endpoints include determination of the recommended MEZI dose plus Kd (Stage 1 only), and assessment of overall survival, overall response rate, time to response, duration of response, time to progression, safety, and quality of life. MeziKd arm Tx consists of 28-day (D) cycles (C) with MEZI on D1–21; 20 mg/m2 intravenous (IV) carfilzomib (CFZ) on D1 of C1, then 56 mg/m2 on D8 and 15 of C1, on D1, 8, and 15 of C2–12, and on D1 and 15 of ≥ C13; and 40 mg oral/IV DEX (20 mg optional in certain pt groups) on D1, 8, 15, and 22. Tx in the Kd arm consists of 28-D cycles with 20 mg/m2 IV CFZ on D1 and 2 of C1, then 56 mg/m2 on D8, 9, 15, and 16 of C1, and on D1, 2, 8, 9, 15, and 16 of ≥ C2; and 20 mg oral/IV DEX (10 mg optional in certain pt groups) on D1, 2, 8, 9, 15, 16, 22, and 23. Tx will continue until progressive disease (PD) or unacceptable toxicity. Key eligibility criteria include age ≥ 18 y, ≥ 1 prior line of anti-MM Tx including LEN and an anti-CD38 monoclonal antibody, minimal response or better to ≥ 1 prior Tx, documented PD during or after last regimen, and no prior CFZ Tx. Enrollment began in October 2022 and is ongoing. Clinical trial information: NCT05552976 .

A pilot study on ultrashort peptide with fluconazole: A promising novel anticandidal combination.

Human infections caused by Candida albicans are common and range in severity from relatively treatable skin and mucosal conditions to systemic, fatal invasive candidiasis. The treatment of fungal infections is challenged by major obstacles, including the scarcity of effective therapeutic options, the toxicity of available medications, and the escalating antifungal resistance. Hence, there exists an urgent need to develop new classes of antimicrobial agents. This study was conducted to investigate the effect of KW-23 peptide against standard and resistant strains of C. albicans alone and in combination with fluconazole. A conjugated ultrashort antimicrobial peptide (KW-23) was designed and synthesized. KW-23 was challenged against standard and multidrug-resistant C. albicans alone and in combination with fluconazole using standard antimicrobial and checkerboard assays. The toxicity of the peptide was examined using hemolytic assays. KW-23 positively affected the standard and resistant Candidal strains (at 5 and 15 μg/mL respectively), exhibiting potent synergistic antimicrobial activity against the standard strain when combined with fluconazole. The effect of the combination was additive against the resistant strain (0.6 μg/mL). Furthermore, the peptide exhibited negligible toxicity on human erythrocytes. KW-23 and its combination with fluconazole could be a promising candidate for developing anticandidal agents.

Discovery of BRD4-HDAC Dual Inhibitors with Improved Fungal Selectivity and Potent Synergistic Antifungal Activity against Fluconazole-Resistant Candida albicans.

Over the past several decades, invasive fungal infections, especially candidiasis, have caused dramatic morbidity and mortality due to ineffective antifungal drugs and severe drug resistance. Herein, new BRD4-histone deacetylase (HDAC) inhibitors were designed to restore the susceptibility of Candida albicans (C. albicans) to fluconazole (FLC). Interestingly, several compounds showed excellent selectivity against fungal HDACs. In particular, compound B2 showed excellent synergistic effect with FLC against resistant C. albicans (FICI = 0.063) with high selectivity against fungal HDACs (SI = 1653) and low cytotoxicity. Compound B2 effectively synergized with FLC and prevented biofilm formation and morphological transition in resistant C. albicans, potentiating the antifungal activity of FLC in vivo and significantly reducing kidney fungal loads. Thus, this drug combination is promising in the treatment of resistant C. albicans infections.

Synergistic anticancer effect of combination treatment of vitamin D and pitavastatin on the HCC1937 breast cancer cells

Vitamin D (Vit D) has anticancer properties including activating cell senescence inhibiting cancer cell proliferation, inducing apoptotic cell death, and decreasing cancer cell migration. On the other hand, statins showed favorable anticancer activities including anti-survival, anti-proliferation, and anti-migration effects. The current study aimed to investigate the synergistic anticancer effect of Vit D and statins against HCC1937 triple-negative breast cancer cells. The antiproliferative effect was tested by MTT assay after 48 hours of the treatments. Trypan blue test and clonogenic assay were used to test the anti-survival activities of the treatments. The ability of the treatments to inhibit the migration ability was tested by scratch assay. Levels of the cell cycle and apoptotic markers were determined by western blotting. Results of the study revealed that all the tested compounds including Vit D, atorvastatin (Ator), simvastatin (Simv), and pitavastatin (Pita) inhibited HCC1937 breast cancer cell growth with different IC50 values ranging from 4.49-12.95 µM. Combined application of Pita and Vit D showed potent synergistic antiproliferative activities against HCC1937 breast cancer cells. The combined therapy of (1µM Vit D and 2 µM Pita) inhibited HCC1937 cell proliferation by cell cycle arrest and apoptosis as evidenced by increasing p21, p53, and cleaved PARP. Finally, the combined treatment decreased the p-STAT3 level in HCC1937 breast cancer cells. The results of the study can be concluded that the combined treatment of Pita and Vit D has a synergistic anticancer effect against HCC1937 breast cancer cells.

Optimization of 1,2,4-Triazole-3-thiones toward Broad-Spectrum Metallo-β-lactamase Inhibitors Showing Potent Synergistic Activity on VIM- and NDM-1-Producing Clinical Isolates.

Metallo-β-lactamases (MBLs) contribute to the resistance of Gram-negative bacteria to carbapenems, last-resort antibiotics at hospital, and MBL inhibitors are urgently needed to preserve these important antibacterial drugs. Here, we describe a series of 1,2,4-triazole-3-thione-based inhibitors displaying an α-amino acid substituent, which amine was mono- or disubstituted by (hetero)aryl groups. Compounds disubstituted by certain nitrogen-containing heterocycles showed submicromolar activities against VIM-type enzymes and strong NDM-1 inhibition (Ki = 10-30 nM). Equilibrium dialysis, native mass spectrometry, isothermal calorimetry (ITC), and X-ray crystallography showed that the compounds inhibited both VIM-2 and NDM-1 at least partially by stripping the catalytic zinc ions. These inhibitors also displayed a very potent synergistic activity with meropenem (16- to 1000-fold minimum inhibitory concentration (MIC) reduction) against VIM-type- and NDM-1-producing ultraresistant clinical isolates, including Enterobacterales and Pseudomonas aeruginosa. Furthermore, selected compounds exhibited no or moderate toxicity toward HeLa cells, favorable absorption, distribution, metabolism, excretion (ADME) properties, and no or modest inhibition of several mammalian metalloenzymes.

Dual Targeting of MDM2 and BCL2/Bclxl Demonstrates Potent Synergistic Activity in High-Risk Adult Acute Lymphoblastic Leukemia

Despite the use of both intensive multi-modal cytotoxic therapeutic regimens and novel immune-based therapies in adult acute lymphoblastic leukemia (ALL) outcomes remain dismal, with five-year survival rates less than 45%. As such, there is an urgent need to identify novel therapies to improve patient outcomes. While TP53 mutations are infrequent in ALL, alternative mechanisms such as MDM2 overexpression or CDKN2A/B deletion may otherwise phenotypically disrupt and circumvent normal p53 function. We previously demonstrated encouraging pre-clinical activity of p53-MDM2 antagonist idasanutlin (RG7388) in diverse high-risk primary and patient-derived xenograft (PDX) ALL samples at concentrations well-below clinically attainable plasma levels [Bell et al., ASH 2020]. However, clinical studies reveal only modest responses to idasanutlin monotherapy in various hematological malignancies, emphasizing the need to identify effective drug combinations. To systematically identify combination treatments which enhance cytotoxic idasanutlin activity, we performed comprehensive high-throughput phenotypic combination screening against 1971 FDA-approved and clinically advanced drugs. Extensive use of an ex vivo coculture system of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSCs) was employed to support growth and survival of the ALL blasts, complemented by high-content image analysis to discriminate and enumerate the respective cell populations. Using the relapsed NALM6 B-ALL cell line and a panel of 6 PDX ALL samples - including relapsed and high-risk cytogenetic subtypes - we iteratively identified drugs (n=10) exhibiting significant synergistic and potent interaction with idasanutlin at concentrations well-below respective drug peak plasma concentrations reported in patients (Bliss synergy δ > 10). These included clinical ALL drugs dexamethasone and daunorubicin, proteasome inhibitor carfilzomib, BCL2/BCLxL inhibitor navitoclax, and histone deacetylase inhibitors romidepsin and pracinostat. Counter-screening of each drug candidate in a NALM6 TP53-isogenic cell model confirmed on-target p53-dependent combination activity (p<0.05). Further interrogation of each drug candidate was performed across a broad landscape of drug combination ratios with idasanutlin in primary (n=5) and PDX samples (n=6), including high-risk KMT2A-rearranged, ABL class T-ALL, and ETP-ALL. Combination of idasanutlin with navitoclax exhibited both the greatest and most consistent synergistic interaction (δ = 24.7±8.7, n=11) of the candidate combinations (n=10) across an extensive landscape of dose combinations; additionally alluding to synergy indiscriminate of ALL subtype. Further characterizing the cytotoxic antileukemic activity of navitoclax, single-agent exposure achieved low nanomolar range, dose-dependent ex vivo sensitivity in a panel of B- (n=10) and T-ALL (n=4) primary and PDX samples, with half maximal inhibitory concentrations (IC50) in the range of 3 to 11 nM (mean IC50 = 6.4 nM) - almost 1000-fold lower than clinically attainable concentrations [Roberts et al., BJH2015]. While both idasanutlin and navitoclax effectively induced apoptosis in their own right, combination exposure markedly enhanced cell death. In an exceptionally high-risk TCF3-HLF PDX sample, 24 hours exposure to the drug combination increased annexin-V positive cells by 37.3±5.0% and 33.3±3.5% as compared to either drug (IC50s, p<0.0001). In the NALM6 and RS4;11 relapsed ALL cell lines, combination exposure significantly increased annexin-V positive cells by at least two-fold (p<0.001) and enhanced apoptotic hallmark products cleaved poly(ADP-ribose) polymerase (PARP) (p<0.01) and cleaved caspase-3 as compared to either drug alone (IC50s). Mechanistically, we found protein levels of MCL-1-binding pro-apoptotic sensitizer NOXA to be up to 3-fold increased in combination-treated blasts (p<0.05), potentially inhibiting compensatory anti-apoptotic MCL-1 activity - a known clinical resistance factor to BH3 mimetics. Together, we provide strong evidence that concurrent targeting of MDM2-p53 binding and BCL2/BCLxL leads to potent and synergistic enhancement of apoptotic cell death in a range of high-risk ALL subtypes. The proposed combination of two clinical-stage compounds could have considerable positive clinical impact for the treatment of adult ALL.

Structure and Activity Relationships of the Two-Component Lantibiotic Bicereucin.

Identified from the pathogen Bacillus cereus SJ1, the two-component lantibiotic bicereucin is featured by the presence of a series of nonproteogenic amino acids and exhibits potent synergistic activity against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci, as well as hemolytic activity against mammalian cells. In this study, we performed site-directed mutagenesis on the nonproteogenic amino acids as well as truncation of dehydrobutyrine-rich N-terminal residues and evaluated the effects on both biological activities. We identified that D-Ala21 and D-Ala26 of Bsjα and D-Ala23 and D-Ala28 of Bsjβ play an essential role in the antimicrobial activity, while the N-termini of both peptides are important for both activities. We also determined that the integrity of both subunits is essential for hemolytic activity. Finally, we obtained two variants BsjαtS17A+Bsjβ and BsjαS30A+BsjβT19A, which retained the antimicrobial activity and exhibited greatly decreased hemolytic toxicity. Overall, our results provide a comprehensive understanding of the structure-activity relationships of bicereucin and insights into the mechanism of action thereof, facilitating the further exploration of the molecular basis of the binding receptor of bicereucin and genome mining of potential novel two-component lantibiotics.

The design and evaluation of the antimicrobial activity of a novel conjugated penta-ultrashort antimicrobial peptide in combination with conventional antibiotics against sensitive and resistant strains of S. aureus and E. coli.

Antimicrobial resistance still constitutes a major health concern to the global human population. The development of new classes of antimicrobial agents is urgently needed to thwart the continuous emergence of highly resistant microbial pathogens. In this study, we have rationally designed a novel conjugated ultrashort antimicrobial peptide. The peptide named naprolyginine was challenged against representative strains of wild-type and multidrug-resistant bacteria individually or in combination with individual antibiotics by employing standard antimicrobial and checkerboard assays. Our results displayed that the peptide exhibits potent synergistic antimicrobial activity against resistant strains of gram-positive and gram-negative bacteria when combined with individual antibiotics. Additionally, the peptide was evaluated for its hemolytic activity against human red blood cells and displayed negligible toxicity. Naprolyginine could prove to be a promising candidate for antimicrobial drug development.

Synthesis and Structure-Activity Studies of β-Barrel Assembly Machine Complex Inhibitor MRL-494.

In the hunt for new antibiotics with activity against Gram-negative pathogens, the outer membrane β-barrel assembly machine (BAM) complex has become an increasingly interesting target. The recently reported BAM complex inhibitor, MRL-494, was discovered via a screening campaign for molecules that target the outer membrane. Notably, MRL-494 was reported to be an unintended byproduct generated during the synthesis of an unrelated compound, and as such no synthesis of the compound was disclosed. We here present a convenient and reliable route for the synthesis of MRL-494 that scales well. The antibacterial activity measured for synthesized MRL-494 matches that reported in the literature. Furthermore, MRL-494 was found to exhibit potent synergistic activity with rifampicin against Gram-negative bacteria, including E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. MRL-494 was also found to cause outer membrane disruption and induction of the Rcs stress response pathway. In addition, we undertook a focused structure-activity study specifically aimed at elucidating the roles played by the two guanidine moieties contained within the structure of MRL-494.

N-acetyl-galactosamine modified metal-organic frameworks to inhibit the growth and pulmonary metastasis of liver cancer stem cells through targeted chemotherapy and starvation therapy

Hepatocellular carcinoma (HCC) is a common high-mortality malignancy which still needs efficient treatments. HCC patients undergoing extrahepatic metastases are mostly with unsatisfactory prognosis. Therefore, specific attention has been paid to extrahepatic HCC metastasis. We integrated Sorafenib (Sor) and glucose oxidase (GOx) into a N-acetyl-galactosamine (GalNAc) modified zeolitic imidazolate framework (ZIF-8), designated as SG@GR-ZIF-8, for targeted bimodal therapies of chemotherapy and starvation therapy against HCC. The hepatic delivery of SG@GR-ZIF was mediated by the specific recognition of GalNAc residues with asialoglycoprotein (ASGPR) on the liver cell surface. Sor is a clinically approved anti-proliferation and anti-angiogenesis drug for advanced HCC treatment. GOx can efficiently induce cell death and disturb malignant progression by suppressing glucose supply of cancer cells, which is highly associated with metabolic rewiring in metastasis. The nano-formulation exhibit significant anti-metastatic HCC activity against C5WN1 cells, a liver cancer stem cell-like cell line with tumorigenicity and pulmonary metastasis activity. In a subcutaneous C5WN1-tumor carrying mouse model, SG@GR-ZIF exhibits potent synergistic anti-tumor activity with a tumor inhibition rate of 89% and a prolonged survival status. The growth and pulmonary metastasis of HCC in an orthotopic mouse model of HCC was remarkably suppressed in SG@GR-ZIF treated group. The therapeutic strategy targeting energy supply combined with first-line treatment holds great promise for the future treatment of metastatic HCC. Statement of significanceSG@GR-ZIF, a N-acetyl-galactosamine modified metal-organic framework carrying Sorafenib and glucose oxidase, was fabricated for treating metastatic hepatocellular carcinoma (HCC). Sorafenib is an anti-proliferation and anti-angiogenesis drug for advanced HCC treatment. Glucose oxidase blocks energy demand in HCC metastasis by depleting glucose. C5WN1 was used for therapeutic evaluations as a liver cancer stem cell-like cell line with tumorigenicity and pulmonary metastasis activity. In subcutaneous C5WN1-tumor bearing mice, SG@GR-ZIF exhibited a tumor inhibition rate of 89% and prolonged survival period. In orthotopic C5WN1-tumor carrying mice, the growth and pulmonary metastasis of hepatocarcinoma was remarkably suppressed by SG@GR-ZIF. Together, this study suggests the great potential of synergistic chemo/starvation therapy mediated by SG@GR-ZIF for treating metastatic HCC.

Determinants of fluconazole resistance and the efficacy of fluconazole and milbemycin oxim combination against Candida parapsilosis clinical isolates from Brazil and Turkey.

Fluconazole-resistant Candida parapsilosis (FLZR-CP) outbreaks are a growing public health concern and have been reported in numerous countries. Patients infected with FLZR-CP isolates show fluconazole therapeutic failure and have a significantly increased mortality rate. Because fluconazole is the most widely used antifungal agent in most regions with outbreaks, it is paramount to restore its antifungal activity. Milbemycin oxim (MOX), a well-known canine endectocide, is a potent efflux pump inhibitor that significantly potentiates the activity of fluconazole against FLZR C. glabrata and C. albicans. However, the FLZ-MOX combination has not been tested against FLZR-CP isolates, nor is it known whether MOX may also potentiate the activity of echinocandins, a different class of antifungal drugs. Furthermore, the extent of involvement of efflux pumps CDR1 and MDR1 and ergosterol biosynthesis enzyme ERG11 and their link with gain-of-function (GOF) mutations in their transcription regulators (TAC1, MRR1, and UPC2) are poorly characterized among FLZR-CP isolates. We analyzed 25 C. parapsilosis isolates collected from outbreaks in Turkey and Brazil by determining the expression levels of CDR1, MDR1, and ERG11, examining the presence of potential GOF mutations in their transcriptional regulators, and assessing the antifungal activity of FLZ-MOX and micafungin-MOX against FLZR and multidrug-resistant (MDR) C. parapsilosis isolates. ERG11 was found to be universally induced by fluconazole in all isolates, while expression of MDR1 was unchanged. Whereas mutations in MRR1 and UPC2 were not detected, CDR1 was overexpressed in three Brazilian FLZR-CP isolates, which also carried a novel TAC1L518F mutation. Of these three isolates, one showed increased basal expression of CDR1, while the other two overexpressed CDR1 only in the presence of fluconazole. Interestingly, MOX showed promising antifungal activity against FLZR isolates, reducing the FLZ MIC 8- to 32-fold. However, the MOX and micafungin combination did not exert activity against an MDR C. parapsilosis isolate. Collectively, our study documents that the mechanisms underpinning FLZR are region specific, where ERG11 mutations were the sole mechanism of FLZR in Turkish FLZR-CP isolates, while simultaneous overexpression of CDR1 was observed in some Brazilian counterparts. Moreover, MOX and fluconazole showed potent synergistic activity, while the MOX-micafungin combination showed no synergy.

PB1850: A DOSE FINDING AND EXPANSION OPEN LABEL, MULTICENTER STUDY OF IADADEMSTAT AND GILTERITINIB IN PATIENTS WITH RELAPSE/REFRACTORY ACUTE MYELOID LEUKEMIA WITH MUTATED FLT3: THE FRIDA STUDY

Background: Despite improvements in therapy for acute myeloid leukemia (AML), relapses remain a common challenge, contributing to the death of more than 50% of AML patients (pts). Approximately a third of pts with newly diagnosed AML harbor mutations in the fms-like tyrosine kinase 3 (FLT3) gene. Current guidelines recommend molecular testing for FLT3-mut at diagnosis and at relapse, for earlier incorporation of targeted agents FLT3 inhibitors (FLT3i). The approval of the FLT3i midostaurin with induction therapy in FLT3mut AML, and of a more specific potent FLT3i, gilteritinib, as monotherapy for relapsed/refractory (R/R) pts have resulted in improved outcomes. Nevertheless, the duration of remission achieved with single-agent gilteritinib in R/R disease is transient and often brief. Iadademstat (iada) is a highly selective and potent covalent inhibitor of LSD1/KDM1A (lysine demethylase 1) that induces differentiation of AML cells in vitro and decreases leukemic stem cell survival in preclinical models. More than 100 subjects have been treated with iada, including R/R AML pts with monotherapy and naïve AML pts in combination with azacitidine, revealing a manageable safety profile and encouraging response rates. The purpose of the FRIDA clinical trial is to evaluate the safety and effect of iada in combination with the standard-of-care gilteritinib for the treatment of R/R FLT3-mut AML pts. The preclinical rationale supporting the combination is based on: 1) a potent synergistic activity in FLT3-AML in several preclinical models, and 2) the anti-leukemic activity of iada, mediated by preventing the interaction between LSD1 and the GFI1 transcription factor and modifying the epigenetic marks of genes resulting on macrophage/monocytic differentiation. These data, together with iada’s favorable ADME and low DDI risk, warrants further exploration of this combination with the aim of improving outcomes for FLT3-mut R/R AML pts. Aims: The FRIDA study will establish the safety, tolerability and determine the recommended phase 2 dose (RP2D), and preliminary activity for the combination of iada and gilteritinib in pts with FLT3mut R/R AML. Methods: Adult pts with a body weight >50Kg and ECOG 0-2, with relapsed or refractory disease, after 1 or 2 prior lines of therapy, and with FLT3-mutations will be enrolled. In a 3 + 3 escalation phase, up to 18 pts would receive iada at doses of 75 to 150 ug, orally, on 5 days ON -2 days OFF schedule, with continuous gilteritinib PO, at 120 mg/day. In the expansion phase, up to 14 pts at the selected safe and pharmacologically active dose/s will be enrolled. Primary endpoints of the study are safety and RP2D determination (based on PK, PD, safety, tolerability, and preliminary activity). Bayesian efficacy monitoring will be performed with cumulating enrollment to ensure futility boundaries are not crossed and to evaluate the activity of the combination. Secondary endpoints include Overall Survival, Event-Free-Survival, Overall Response Rate, Time to Response, Duration of Response and Transfusion rate. Exploratory endpoints include measurable residual disease and gene mutational analysis. Results: This is a clinical trial in progress. No results available. Summary/Conclusion: Study recruiting in Q2 2022 in US.

P610: SYNERGISTIC ACTIVITY OF FTO INHIBITOR FB23-2 WITH IBRUTINIB IN XENOGRAFT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Several studies have documented that FB23-2, as the selective inhibitor of RNA N6-methyladenosine (m6A) demethylase FTO, exerts crucial role in dampening tumorigenesis. Indeed, FTO deactivation was proved to render resistant leukemia cells sensitive to targeted agents, which suggests a potential combination therapy strategy of FB23-2. Aims: The present study was aimed to identify the synergistic utility of FB23-2 combination with ibrutinib in CLL. Methods: A disseminated leukemia xenograft murine model was established to explore the efficacy of FB23-2 in CLL. Leukemia burden of CLL mice was quantified by luciferase intensity using bioluminescence imaging. Bone marrow and spleen infiltration of leukemic cells was assessed by flow cytometry. And cell survival was determined by cell counting kit-8. Results: To investigate the combinatory effect of FB23-2 and ibrutinib, cell viability assay was performed firstly. FB23-2 displayed potent synergistic activity with ibrutinib in promoting cytotoxicity in CLL cells (Figure 1A). Consistently, FTO silencing mediated by lentivirus exhibited exquisite sensitivity to ibrutinib in CLL cells (Figure 1B). Besides, CLL primary cells and MEC-1 cells with treatment of FB23-2 and ibrutinib simultaneously triggered increased cell apoptosis compared to single agent (Figure 1C). Further underlying cooperative mechanism investigation demonstrated an aberrant activation of DNA damage pathway after serial dilution of FB23-2 treatment. Hence, enhanced expression of p-ATM, p-chk2 and p-H2AX was detected in FTO knockdown and ibrutinib co-treatment group, indicating FTO inhibition inducing ibrutinib-sensitivity via regulating DNA damage (Figure 1D). In addition, we investigated the efficacy of FB23-2 alone and corporation in CLL xenograft murine model. Compared to ibrutinib alone, combination treatment with FB23-2 significantly prolonged the lifespan of CLL mice (median survival time 20 versus 22.5 days, respectively, p=0.02, Figure 1E). FB23-2+ibrutinib group showed an obviously declined proportion of CLL cells compared to ibrutinib-treated alone both in bone marrow(20.93%±2.95% versus 37.17%±4.22%, p=0.02) and spleen (23.58%±6.03% and 54.58%±9.01%, p=0.03, Figure 1F-G). Similar results were obtained with three treated group relative to control group (all p<0.05). Moreover, FB23-2 combined with ibrutinib diminished leukemia burden and splenomegaly in CLL mice (Figure 1H-I). As shown in Figure 1J, leukemia involvement and malignant proliferation were more evident in control group, confirming the potent anti-leukemic activity of FB23-2. Image:Summary/Conclusion: Taken together, our investigation provides pre-clinical evidence for the utility of FB23-2 in CLL, especially combinatory benefit with ibrutinib. FB23-2 represents a promising strategy to novel treatment optimum in CLL.

Exploring the significance of nanomaterials and organic amendments — Prospect for phytoremediation of contaminated agroecosystem

Emerging micro-pollutants have rapidly contaminated the agro-ecosystem, posing serious challenges to a sustainable future. The vast majority of them have infiltrated the soil and damaged agricultural fields and crops after being released from industry. These pollutants and their transformed products are also transported in vast quantities which further exacerbate the damage. Sustainable remediation techniques are warranted for such large amounts of contaminants. As aforementioned, many of them have been detected at very high concentrations in soil and water which adversely affect crop physiology by disrupting different metabolic processes. To combat this situation, nanomaterials and other organic amendments assisted phytoremediation ware considered as a viable alternative. It is a potent synergistic activity between the biological system and the supplied organic or nanomaterial material to eliminate emerging contaminants and micropollutants from crop fields. This can be effectively be applied to degraded crop fields and could potentially embody a green technology for sustainable agriculture.

Synergistic antimicrobial activity of TZP4 with conventional antibiotics against antibiotic‐resistant Pseudomonas aeruginosa

AbstractWe recently synthesized a triazine‐derived amphipathic molecule, TZP4, which has potent antimicrobial activity against antibiotic‐resistant Pseudomonas aeruginosa (ARPA). Here, we investigated the synergistic antimicrobial effect of TZP4 in combination with four different conventional antibiotics each representing a distinct mechanism against ARPA. Checkerboard assay and time‐kill assays showed that the potent synergistic antimicrobial activity was observed only in TZP4/chloramphenicol and TZP4/ciprofloxacin combinations. Flow cytometry analysis for these combinations demonstrated that the synergistic antimicrobial activity was due to the increased permeability of bacterial cytoplasmic membrane. In addition, TZP4 was able to prevent and eradicate biofilms, which are one of the primary reasons for drug resistance caused by ARPA. Confocal laser scanning microscopy revealed that TZP4 significantly reduced mature ARPA biofilm biomass. Overall, our findings suggest that TZP4 is a potential adjuvant to antimicrobial chemotherapy and a potential antibiofilm agent to treat infections caused by ARPA.