Combining MEK and SRC inhibitors for treatment of colorectal cancer demonstrate increased efficacy in vitro but not in vivo.

Metastatic colorectal cancer (mCRC) is the second leading cause of cancer deaths in the United States. More than 50% of patients with mCRC harbor mutations of the oncogenic driver RAS (KRAS or NRAS). Because directly targeting most mutations of RAS is technically challenging, researchers have concentrated on targeting MEK, a downstream mediator of RAS. However, targeting MEK as single-agent therapy is ineffective in patients with mCRC. We hypothesize that combining a MEK inhibitor with other agents can enhance the efficacy of MEK targeting in mCRC. Unbiased high-throughput screening (HTS) was performed to identify drugs that enhance the efficacy of MEK inhibitors. HTS was performed with KRAS-mutated CRC cells using the MEK inhibitor trametinib as a "backbone" and two "clinically ready" compound libraries approved by the U.S. Food and Drug Administration or in clinical trials. HTS demonstrated that the combination of the SRC inhibitor dasatinib and trametinib was synergistic in CRC cells in vitro (MTT and colony formation assays). Analysis of markers for cell proliferation and apoptosis using fluorescence-activated cell sorting, reverse-phase protein array, or Western blotting demonstrated decreased cell proliferation and increased cell death when targeting both SRC and MEK as compared to single agents in multiple CRC cell lines. However, combining dasatinib and trametinib in vivo at doses in mice equivalent to doses used in humans failed to significantly enhance the antitumor activity of trametinib when compared to that of trametinib alone. These results underscore the importance of performing careful preclinical in vivo validation studies using clinically relevant doses as a prerequisite for translating in vitro findings to the clinic.

Rivaroxaban and apixaban are less effective than enoxaparin for the prevention of catheter-induced clotting in vitro.

BACKGROUND: Central venous catheters are prone to clotting, particularly in patients with cancer. Although low-molecular-weight heparin and direct oral anticoagulants, such as apixaban and rivaroxaban, have been evaluated for the prevention of catheter thrombosis, their efficacy remains uncertain. OBJECTIVES: Compare apixaban and rivaroxaban with enoxaparin for the prevention of catheter-induced clotting in vitro. METHODS: To address this uncertainty, we used a well-established microplate-based assay to compare the effects of enoxaparin, apixaban, and rivaroxaban on catheter-induced thrombosis and thrombin generation in human plasma. RESULTS: Consistent with our previous findings, catheter segments shortened the clotting time and promoted thrombin generation. When compared at concentrations with similar anti-factor Xa activity as enoxaparin, apixaban and rivaroxaban were >20-fold less potent than enoxaparin for the prevention of catheter-induced clotting and thrombin generation. CONCLUSION: The prevention of catheter thrombosis in patients with cancer is challenging. Clinical trials are needed to compare the efficacy of low-molecular-weight heparin with that of direct oral anticoagulants both for the prevention and treatment of catheter thrombosis.

Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential.

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.

Discovery of a Novel Covalent EZH2 Inhibitor Based on Tazemetostat Scaffold for the Treatment of Ovarian Cancer.

Enhancer of zeste homologue 2 (EZH2) is the enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2), which plays an important role in post-translational modifications of histones. In this study, we designed and synthesized a new series EZH2 covalent inhibitors that have rarely been reported. Biochemical studies and mass spectrometry provide information that SKLB-03220 could covalently bind to the S-adenosylmethionine (SAM) pocket of EZH2. Besides, SKLB-03220 was highly potent for EZH2MUT, while exhibiting weak activities against other tested histone methyltransferases (HMTs) and kinases. Moreover, SKLB-03220 displayed noteworthy potency against ovarian cancer cell lines and continuously abolished H3K27me3 after washing out. Furthermore, oral administration of SKLB-03220 significantly inhibited tumor growth in PA-1 xenograft model without obvious adverse effects. Taken together, SKLB-03220 is a potent, selective EZH2 covalent inhibitor with noteworthy anticancer efficacy both in vitro and in vivo.

Efficacy of Dabrafenib and Trametinib in a Patient with Squamous-Cell Carcinoma, with Mutation p.D594G in BRAF and p.R461* in NF1 Genes-A Case Report with Literature Review.

The 3rd class of BRAF (B-Raf Proto-Oncogene, Serine/Threonine Kinase) variants including G466, D594, and A581 mutations cause kinase death or impaired kinase activity. It is unlikely that RAF (Raf Proto-Oncogene, Serine/Threonine Kinase) inhibitors suppress ERK (Extracellular Signal-Regulated Kinase) signaling in class 3 mutant-driven tumors due to the fact that they preferentially inhibit activated BRAF V600 mutants. However, there are suggestions that class 3 mutations are still associated with enhanced RAS/MAPK (RAS Proto-Oncogene, GTPase/Mitogen-Activated Protein Kinase) activation, potentially due to other mechanisms such as the activation of growth factor signaling or concurrent MAPK pathway mutations, e.g., RAS or NF1 (Neurofibromin 1). A 75-year-old male patient with squamous-cell cancer (SqCC) of the lung and with metastases to the kidney and mediastinal lymph nodes received chemoimmunotherapy (expression of Programmed Cell Death 1 Ligand 1 (PD-L1) on 2% of tumor cells). The chemotherapy was limited due to the accompanying myelodysplastic syndrome (MDS), and pembrolizumab monotherapy was continued for up to seven cycles. At the time of progression, next-generation sequencing was performed and a c.1781A>G (p.Asp594Gly) mutation in the BRAF gene, a c.1381C>T (p.Arg461Ter) mutation in the NF1 gene, and a c.37C>T (p.Gln13Ter) mutation in the FANCC gene were identified. Combined therapy with BRAF (dabrafenib) and MEK (trametinib) inhibitors was used, which resulted in the achievement of partial remission of the primary lesion and lung nodules and the stabilization of metastatic lesions in the kidney and bones. The therapy was discontinued after five months due to myelosuppression associated with MDS. The molecular background was decisive for the patient's fate. NSCLC patients with non-V600 mutations in the BRAF gene rarely respond to anti-BRAF and anti-MEK therapy. The achieved effectiveness of the treatment could be related to a mutation in the NF1 tumor suppressor gene. The loss of NF1 function causes the excessive activation of KRAS and overactivity of the signaling pathway containing BRAF and MEK, which were the targets of the therapy. Moreover, the mutation in the FANCC gene was probably related to MDS development. The NGS technique was crucial for the qualification to treatment and the prediction of the NSCLC course in our patient. The mutations in two genes­the BRAF oncogene and the NF1 tumor suppressor gene­were the reason for the use of dabrafenib and trametinib treatment. The patients achieved short-term disease stabilization. This proved that coexisting mutations in these genes affect the disease course and treatment efficacy.

Primary resistance to integrase inhibitors in Shenzhen.

OBJECTIVES: In recent years, integrase strand transfer inhibitor (INSTI)-containing regimens have been increasingly adopted in treatment for HIV/AIDS and promoted as non-occupational post-exposure prophylaxis in China. This study aims to describe the prevalence of resistance to integrase and drug resistance mutations (DRMs) among ART-naive patients in Shenzhen, China. METHODS: Serum samples and demographic information were collected from newly reported ART-naive patients in Shenzhen in 2020. The study sequenced the coding sequence of the HIV-1 integrase gene and determined the DRMs.​. RESULTS: In this study, 1682 newly reported cases were included and 1071 of them were successfully sequenced finally. The prevalence of primary drug resistance was 1.77%, with 19 samples showing varying degrees of resistance to INSTIs. The study detected six major DRMs in 16 individuals and eight accessory DRMs in 24 individuals. The prevalence of transmitted drug resistance (TDR) mutations was 1.21%, with five transmitted mutations detected in 13 individuals. The prevalence of drug resistance to raltegravir and elvitegravir was statistically higher than to bictegravir, cabotegravir and dolutegravir. CONCLUSIONS: The prevalence of INSTI resistance in Shenzhen in 2020 was relatively high. ​Continued surveillance for resistance to INSTIs is recommended and treatment regimens should be adopted based on the pattern of resistance to INSTIs. ​Dolutegravir or bictegravir is first recommended when considering INSTIs as treatment regimens.

In vivo pharmacokinetic and pharmacodynamic study of co-spray-dried inhalable pirfenidone microparticles in rats.

Pirfenidone (PRF) is the first FDA-approved API in the treatment of idiopathic pulmonary fibrosis (IPF). However, PRF induces serious side effects, such as photophobia and gastrointestinal disorder. PRF inhalation can be expected with a lower effective dose and reduced side effects. In this study, PRF was prepared as inhalable co-spray-dried particles for dry powder inhalation. Mannitol, L-leucine (Leu), and NaCl were used as a stabilizer. The kinds and ratios of stabilizers affecting the physicochemical properties of particles were analyzed, including particle size and surface composition, because of the surface enrichment properties of Leu, the most effective stabilizer. The co-spray-dried PRF and Leu microparticle (SD-PL1:1) have the smallest size and highest aerosol performance. The bioavailability was confirmed by in vivo pharmacokinetics (PK) studies. In addition, in vivo pharmacodynamics (PD) experiments were conducted using a bleomycin-induced IPF rat model. In vivo PK experiments demonstrated that pulmonary administration of SD-PL1:1 was 4 times more effective than the oral route. Similar to the PK results, the therapeutic effect was improved when SD-PL1:1 was administered via the pulmonary route compared to the oral route.

Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Impact of body weight change on clinical outcomes in patients with idiopathic pulmonary fibrosis receiving pirfenidone.

There have been limited studies on the association between prognosis and body weight change in patients with idiopathic pulmonary fibrosis (IPF). This single-center retrospective observational study evaluated the impact of weight loss on outcomes in Korean patients with IPF receiving pirfenidone at a tertiary medical institution. We analyzed 215 IPF patients prescribed pirfenidone from January 1st, 2015 to December 31st, 2019. The patients were categorized into maintained weight (MW; weight gain or loss < 5%/year) and reduced weight (RW; weight loss ≥ 5%/year) groups. The mean age was 71.8 years and 175 (81.4%) were male. There were 54 (25.1%) patients in the RW group. All patients showed a decrease in body weight (baseline vs. after 1 year; 64.1 kg vs. 62.8 kg, P < 0.001). Although baseline lung function showed a difference, there was no difference in the rate of change (forced vital capacity [% of predicted]; P = 0.221, diffusing capacity of the lung for carbon monoxide [% of predicted]; P = 0.973). The MW group had a lower risk of all-cause mortality (P < 0.001). Weight loss appeared to be a significant risk factor for mortality in patients with IPF. Not only disease control with antifibrotic agents, but also efforts to prevent weight loss may be necessary.

Investigating the possible mechanisms of pirfenidone to be targeted as a promising anti-inflammatory, anti-fibrotic, anti-oxidant, anti-apoptotic, anti-tumor, and/or anti-SARS-CoV-2.

Pirfenidone (PFD) is a non-peptide synthetic chemical that inhibits the production of transforming growth factor-beta 1 (TGF-ß1), tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF), Interleukin 1 beta (IL-1ß), and collagen 1 (COL1A1), all of which have been linked to the prevention or removal of excessive scar tissue deposition in many organs. PFD has been demonstrated to decrease apoptosis, downregulate angiotensin-converting enzyme (ACE) receptor expression, reduce inflammation through many routes, and alleviate oxidative stress in pneumocytes and other cells while protecting them from COVID-19 invasion and cytokine storm. Based on the mechanism of action of PFD and the known pathophysiology of COVID-19, it was recommended to treat COVID-19 patients. The use of PFD as a treatment for a range of disorders is currently being studied, with an emphasis on outcomes related to reduced inflammation and fibrogenesis. As a result, rather than exploring the molecule's chemical characteristics, this review focuses on innovative PFD efficacy data. Briefly, herein we tried to investigate, discuss, and illustrate the possible mechanisms of actions for PFD to be targeted as a promising anti-inflammatory, anti-fibrotic, anti-oxidant, anti-apoptotic, anti-tumor, and/or anti-SARS-CoV-2 candidate.

Genotypic correlates of resistance to the HIV-1 strand transfer integrase inhibitor cabotegravir.

Cabotegravir (CAB) is an integrase strand transfer inhibitor (INSTI) formulated as a long-acting injectable drug approved for pre-exposure prophylaxis and use with a long acting rilpivirine formulation for therapy in patients with virological suppression. However, there has been no comprehensive review of the genetic mechanisms of CAB resistance. Studies reporting the selection of drug resistance mutations (DRMs) by CAB and the results of in vitro CAB susceptibility testing were reviewed. The impact of integrase mutations on CAB susceptibility was assessed using regularized regression analysis. The most commonly selected mutations in the 24 persons developing virological failure while receiving CAB included Q148R (n = 15), N155H (n = 7), and E138K (n = 5). T97A, G118R, G140 A/R/S, and R263K each developed in 1-2 persons. With the exception of T97A, G118R, and G140 A/R, these DRMs were also selected in vitro while G140R was selected in the SIV macaque model. Although these DRMs are similar to those occurring in persons receiving the related INSTI dolutegravir, Q148R was more likely to occur with CAB while G118R and R263K were more likely to occur with dolutegravir. Regularized regression analysis identified 14 DRMs significantly associated with reduced CAB susceptibility including six primary DRMs which reduced susceptibility on their own including G118R, Q148 H/K/R, N155H, and R263K, and eight accessory DRMs including M50I, L74 F/M, T97A, E138K, and G140 A/C/S. Isolates with Q148 H/K/R in combination with L74M, E138 A/K, G140 A/S, and N155H often had >10-fold reduced CAB susceptibility. M50I, L74M, and T97A are polymorphic mutations that alone did not appear to increase the risk of virological failure in persons receiving a CAB-containing regimen. Careful patient screening is required to prevent CAB from being used during active virus replication. Close virological monitoring is required to minimize CAB exposure to active replication to prevent the emergence of DRMs associated with cross-resistance to other INSTIs.

Plasticity towards Rigidity: A Macrophage Conundrum in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and ultimately fatal diffuse parenchymal lung disease. The molecular mechanisms of fibrosis in IPF patients are not fully understood and there is a lack of effective treatments. For decades, different types of drugs such as immunosuppressants and antioxidants have been tested, usually with unsuccessful results. Although two antifibrotic drugs (Nintedanib and Pirfenidone) are approved and used for the treatment of IPF, side effects are common, and they only slow down disease progression without improving patients' survival. Macrophages are central to lung homeostasis, wound healing, and injury. Depending on the stimulus in the microenvironment, macrophages may contribute to fibrosis, but also, they may play a role in the amelioration of fibrosis. In this review, we explore the role of macrophages in IPF in relation to the fibrotic processes, epithelial-mesenchymal transition (EMT), and their crosstalk with resident and recruited cells and we emphasized the importance of macrophages in finding new treatments.

Survival and lung function decline in patients with definite, probable and possible idiopathic pulmonary fibrosis treated with pirfenidone.

BACKGROUND: There is no clear evidence whether pirfenidone has a benefit in patients with probable or possible UIP, i.e. when idiopathic pulmonary fibrosis (IPF) is diagnosed with a lower degree of diagnostic certainty. We report on outcomes of treatment with pirfenidone in IPF patients diagnosed with various degrees of certainty. METHODS AND FINDINGS: We followed patients in the multi-national European MultiPartner IPF Registry (EMPIRE) first seen between 2015 and 2018. Patients were assessed with HRCT, histopathology and received a multi-disciplinary team (MDT) IPF diagnosis. Endpoints of interest were overall survival (OS), progression-free survival (PFS) and lung function decline. RESULTS: A total of 1626 patients were analysed, treated with either pirfenidone (N = 808) or receiving no antifibrotic treatment (N = 818). When patients treated with pirfenidone were compared to patients not receiving antifibrotic treatment, OS (one-, two- and three-year probability of survival 0.871 vs 0.798; 0.728 vs 0.632; 0.579 vs 0.556, P = 0.002), and PFS (one-, two- and three-year probability of survival 0.597 vs 0.536; 0.309 vs 0.281; 0.158 vs 0.148, P = 0.043) was higher, and FVC decline smaller (-0.073 l/yr vs -0.169 l/yr, P = 0.017). The benefit of pirfenidone on OS and PFS was also seen in patients with probable or possible IPF. CONCLUSIONS: This EMPIRE analysis confirms the favourable outcomes observed for pirfenidone treatment in patients with definitive IPF and indicates benefits also for patients with probable or possible IPF.

Alleviation of cardiac fibrosis using acellular peritoneal matrix-loaded pirfenidone nanodroplets after myocardial infarction in rats.

Myocardial fibrosis (MF) in the remote myocardium is a feature at the micoscopic level of pathological remodeling after myocardial infarction (MI). Although pirfenidone (PFD), an antifibrotic agent, is commonly used to inhibit fibrosis in multiple organs, its clinical use is limited because of the high doses required for favorable therapeutic outcomes and various side effects. Nanodrug technology has allowed for delayed quantitative drug release and reduced the amount of medication required, improving the treatment strategy for MF. In this study, we investigated the possible therapeutic effect of peritoneal matrix-loaded pirfenidone nanodroplets (NDs) on MI fibrosis. The results showed that the Perfluoropentane-Pirfenidone@Nanodroplets-Polyethylene glycol 2000 (PFP-PFD@NDs-PEG) described in this study was successfully synthesized and demonstrated a high potential for the targeted treatment of MI. The total duration of pirfenidone release from PFP-PFD@NDs-PEG was increased by loading it into an acellular peritoneal matrix (APM). Additionally, pirfenidone inhibited the transformation of cardiac fibroblasts into cardiac myofibroblasts in vitro and reduced the synthesis and secretion of collagen I and collagen III by cardiac myofibroblasts. The combination of the APM with pirfenidone nanodroplets achieved a slow drug release and showed excellent therapeutic effects on fibrosis in MI rats. Our study confirmed the feasibility and synergistic effectiveness of the APM combined with pirfenidone nanodroplets in the treatment of fibrosis in MI rats. Moreover, our technique offers a great potential for applying nanomedicine in other biomedical fields.

Application of Activators Ecarin and Factor Xa in Thrombelastography for Measurement of Anticoagulant Effect of Direct Oral Anticoagulants Using TEG 5000.

There are situations where monitoring direct oral anticoagulants (DOACs) would be useful, including bleedings and trauma. The thromboelastographic technique has proven useful in bleeding situations in trauma and heart surgery. The aim of this study was to examine the effect of DOACs on all currently commercially available conventional TEG®5000 assays as well as novel modified assay using Ecarin and human factor Xa (HFXa). Healthy male volunteers were given single dose of oral dabigatran 150 mg, rivaroxaban 20 mg, or apixaban 5 mg. Kaolin, RapidTEG, functional fibrinogen, PlateletMapping assay, and novel modified assays using Ecarin and HFXa were prepared. All TEG parameters were recorded. DOAC concentrations were correlated to the parameters with highest response to the DOAC effect. Sensitivity and negative predictive value of the parameter with highest response to DOAC concentration of 50 ng/mL was calculated. None of the conventional TEG assays demonstrated significant response to the effect on apixaban. Using Ecarin, reaction time R was strongly correlated with dabigatran concentrations. Using HFXa assay, R was strongly correlated with rivaroxaban and apixaban concentrations: r = 0.96, 0.84, and 0.86, respectively; p < 0.0001 for all. The R times obtained with the modified assays demonstrated strong sensitivity and negative predictive values for DOAC levels of ≥50 ng/mL. We have demonstrated that TEG®5000 can monitor the DOAC effect on hemostasis when the appropriate activator is used with significant correlation with DOAC concentrations. Larger clinical studies are warranted for correlation of TEG profile and clinical outcomes.

Integrase inhibitors in children and adolescents: clinical use and resistance.

BACKGROUND: Although integrase inhibitor (INI)-based regimens are now the first-line choice for all people living with HIV, experience among children and adolescents is still scarce. We describe the characteristics and outcomes of a paediatric/adolescent cohort on INI-based ART. METHODS: Retrospective analysis of HIV-infected patients below 18 years of age who started an INI-based regimen from 2007 to 2019, enrolled in the Spanish National Adult (CoRIS) and Paediatric (CoRISpe) cohorts. Resistance mutations were identified by the Stanford HIV Drug Resistance Database. RESULTS: Overall, 318 INI-based regimens were implemented in 288 patients [53.8% female; median age at start of 14.3 years (IQR 12.0-16.3)]. Most were born in Spain (69.1%), vertically infected (87.7%) and treatment-experienced (92.7%). The most frequently prescribed INI was dolutegravir (134; 42.1%), followed by raltegravir (110; 34.6%) and elvitegravir (73; 23.0%). The median exposure was 2.0 years (IQR 1.1-3.0). The main reasons to start an INI-based therapy were treatment simplification (54.4%) and virological failure (34.3%). In total, 103 (32.4%) patients interrupted their regimen: 14.5% for simplification and 8.5% due to virological failure. Most subjects who received dolutegravir (85.8%) and elvitegravir (83.6%) did not interrupt their regimen and maintained undetectable viral load. There were only five virological failures with dolutegravir and three with elvitegravir. There were no interruptions related to adverse events. Seven patients with virological failure presented major resistance mutations to INIs; none of them were on dolutegravir. CONCLUSIONS: INI-based regimens were effective and safe for HIV treatment in children and adolescents. Dolutegravir and elvitegravir presented an excellent profile, and most patients achieved and maintained viral suppression.

New Deferric Amine Compounds Efficiently Chelate Excess Iron to Treat Iron Overload Disorders and to Prevent Ferroptosis.

Excess iron accumulation occurs in organs of patients with certain genetic disorders or after repeated transfusions. No physiological mechanism is available to excrete excess iron and iron overload to promote lipid peroxidation to induce ferroptosis, thus iron chelation becomes critical for preventing ion toxicity in these patients. To date, several iron chelators have been approved for iron chelation therapy, such as deferiprone and deferoxamine, but the current iron chelators suffer from significant limitations. In this context, new agents are continuously sought. Here, a library of new deferric amine compounds (DFAs) with adjustable skeleton and flexibility is synthesized by adopting the beneficial properties of conventional chelators. After careful evaluations, compound DFA1 is found to have greater efficacy in binding iron through two molecular oxygens in the phenolic hydroxyl group and the nitrogen atom in the amine with a 2:1 stoichiometry. This compound remarkably ameliorates iron overload in diverse murine models through both oral and intravenous administration, including hemochromatosis, high iron diet-induced, and iron dextran-stimulated iron accumulation. Strikingly, this compound is found to suppress iron-induced ferroptosis by modulating the intracellular signaling that drives lipid peroxidation. This study opens a new approach for the development of iron chelators to treat iron overload.

Effect of Baloxavir and Oseltamivir in Combination on Infection with Influenza Viruses with PA/I38T or PA/E23K Substitutions in the Ferret Model.

Amino acid substitutions I38T and E23K in the influenza polymerase acidic (PA) protein lead to reduced susceptibility to the influenza antiviral drug baloxavir. The in vivo effectiveness of baloxavir and oseltamivir for treatment of these viruses is currently unknown. Using patient-derived influenza isolates, combination therapy was equally effective as monotherapy in reducing viral titers in the upper respiratory tract of ferrets infected with A(H1N1pdm09)-PA/E23K or A(H3N2)-PA/I38T. When treated with baloxavir plus oseltamivir, infection with a mixture of PA/I38T or PA/E23K and corresponding wild-type virus was characterized by a lower selection of viruses with reduced baloxavir susceptibility over the course of infection compared to baloxavir monotherapy. De novo emergence of the oseltamivir resistance mutation NA/H275Y occurred in ferrets treated with oseltamivir alone but not in ferrets treated with baloxavir plus oseltamivir. Our data suggest that combination therapy with influenza drugs with different mechanisms of action decreased the selection pressure for viruses with reduced drug susceptibility. IMPORTANCE Influenza viruses cause significant morbidity and mortality worldwide but can be treated with antiviral drugs. In 2018, a highly effective antiviral drug, baloxavir marboxil, was licensed. However, the selection of viruses with baloxavir resistance was relatively high following treatment, which may compromise the effectiveness of the drug. Here, we took two different influenza viruses that are resistant to baloxavir and tested the effectiveness alone and in combination with oseltamivir (a second influenza antiviral drug) in the ferret model. Our findings suggest that combination treatment may be a more effective method than monotherapy to reduce the selection of resistant viruses. These results may have important clinical implications for the treatment of influenza.

Mivebresib synergized with PZ703b, a novel Bcl-xl PROTAC degrader, induces apoptosis in bladder cancer cells via the mitochondrial pathway.

Bladder cancer is a common urinary cancer that still lacks effective treatments. In the present study, we evaluated the effect of BET inhibitor, mivebresib, in combination with PZ703b, a Bcl-xl PROTAC, on apoptosis in bladder cancer cells. The results revealed that mivebresib and PZ703b synergistically decreased the viabilities of bladder cancer cells. Co-treatment of mivebresib and PZ703b induced apoptosis in bladder cancer cells via the mitochondrial pathway in a caspase-dependent manner. Mechanistically, mivebresib and PZ703b treatment inhibited the expression of Mcl-1 and Bcl-xl, accompanied by upregulation of Bim. Hence, co-treatment of mivebresib and PZ703b rebalanced the level of pro- and anti-apoptotic Bcl-2 proteins in cells. Further investigations showed that forced expression of Mcl-1 or Bcl-xl markedly protected bladder cancer cells from apoptosis induced by combination treatment of mivebresib and PZ703b. In addition, knockdown of Bim also inhibited the cell death induced by mivebresib/PZ703b in bladder cancer cells. In summary, our findings reveal that the combination treatment of mivebresib and PZ703b represents a novel promising strategy to treat bladder cancer.

Cryo-EM structures of wild-type and E138K/M184I mutant HIV-1 RT/DNA complexed with inhibitors doravirine and rilpivirine.

Structures trapping a variety of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in explaining the mechanisms of catalysis, inhibition, and drug resistance and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-electron microscopy (cryo-EM) has been challenging due to the enzyme's relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and differ from the typical conformation of nonnucleoside RT inhibitor (NNRTI)-bound RT/double-stranded DNA (dsDNA), RT/RNA-DNA, and RT/dsRNA complexes; the primer grip in RT/DNA/doravirine and the YMDD motif in RT/DNA/rilpivirine have large shifts. The DNA primer 3'-end in the doravirine-bound structure is positioned at the active site, but the complex is in a nonproductive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation opens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by an earlier kinetic study. These structural differences have implications for understanding molecular mechanisms of drug resistance and for drug design.