Pharmacological Resistance Research Articles

Bioinformatic Analysis from a Descriptive Profile of miRNAs in Chronic Migraine.

Chronic migraines have been described chiefly only from a clinical perspective. However, searching for reliable molecular markers has allowed for the discovery of the expression of different genes mainly associated with inflammation, neuro-vascularization, and pain-related pathways. The interest in microRNAs (miRs) that can regulate the expression of these genes has gained significant relevance since multiple miRs could play a key role in regulating these events. In this study, miRs were searched in samples from patients with chronic migraine, and the inclusion criteria were carefully reviewed. Different bioinformatic tools, such as miRbase, targetscan, miRPath, tissue atlas, and miR2Disease, were used to analyze the samples. Our findings revealed that some of the miRs were expressed more (miR-197, miR-101, miR-92a, miR-375, and miR-146b) and less (miR-133a/b, miR-134, miR-195, and miR-340) than others. We concluded that, during chronic migraine, common pathways, such as inflammation, vascularization, neurodevelopment, nociceptive pain, and pharmacological resistance, were associated with this disease.

Autophagy modulation attenuates sorafenib resistance in HCC induced in rats

Hepatocellular carcinoma (HCC) has risen as the villain of cancer-related death globally, with a usual cruel forecasting. Sorafenib was officially approved by the FDA as first-line treatment for advanced HCC. Despite the brilliant promise revealed in research, actual clinical results are limited due to the widespread appearance of drug resistance. The tumor microenvironment (TME) has been correlated to pharmacological resistance, implying that existing cellular level strategies may be insufficient to improve therapy success. The role of autophagy in cancer is a two-edged sword. On one hand, autophagy permits malignant cells to overcome stress, such as hypoxic TME and therapy-induced starvation. Autophagy, on the other hand, plays an important role in damage suppression, which can reduce carcinogenesis. As a result, controlling autophagy is certainly a viable technique in cancer therapy. The goal of this study was to investigate at the impact of autophagy manipulation with sorafenib therapy by analyzing autophagy induction and inhibition to sorafenib monotherapy in rats with HCC. Western blot, ELISA, immunohistochemistry, flow cytometry, and quantitative-PCR were used to investigate autophagy, apoptosis, and the cell cycle. Routine biochemical and pathological testing was performed. Ultracellular features and autophagic entities were observed using a transmission electron microscope (TEM). Both regimens demonstrated significant reductions in chemotherapeutic resistance and hepatoprotective effects. According to the findings, both autophagic inhibitors and inducers are attractive candidates for combating sorafenib-induced resistance in HCC.

S-allyl-cysteine triggers cytotoxic events in rat glioblastoma RG2 and C6 cells and improves the effect of temozolomide through the regulation of oxidative responses

Glioblastoma (GBM) is an aggressive form of cancer affecting the Central Nervous System (CNS) of thousands of people every year. Redox alterations have been shown to play a key role in the development and progression of these tumors as Reactive Oxygen Species (ROS) formation is involved in the modulation of several signaling pathways, transcription factors, and cytokine formation. The second-generation oral alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic drug used to treat of GBM, though patients often develop primary and secondary resistance, reducing its efficacy. Antioxidants represent promising and potential coadjutant agents as they can reduce excessive ROS formation derived from chemo- and radiotherapy, while decreasing pharmacological resistance. S-allyl-cysteine (SAC) has been shown to inhibit the proliferation of several types of cancer cells, though its precise antiproliferative mechanisms remain poorly investigated. To date, SAC effects have been poorly explored in GBM cells. Here, we investigated the effects of SAC in vitro, either alone or in combination with TMZ, on several toxic and modulatory endpoints—including oxidative stress markers and transcriptional regulation—in two glioblastoma cell lines from rats, RG2 and C6, to elucidate some of the biochemical and cellular mechanisms underlying its antiproliferative properties. SAC (1–750 µM) decreased cell viability in both cell lines in a concentration-dependent manner, although C6 cells were more resistant to SAC at several of the tested concentrations. TMZ also produced a concentration-dependent effect, decreasing cell viability of both cell lines. In combination, SAC (1 µM or 100 µM) and TMZ (500 µM) enhanced the effects of each other. SAC also augmented the lipoperoxidative effect of TMZ and reduced cell antioxidant resistance in both cell lines by decreasing the TMZ-induced increase in the GSH/GSSG ratio. In RG2 and C6 cells, SAC per se had no effect on Nrf2/ARE binding activity, while in RG2 cells TMZ and the combination of SAC + TMZ decreased this activity. Our results demonstrate that SAC, alone or in combination with TMZ, exerts antitumor effects mediated by regulatory mechanisms of redox activity responses. SAC is also a safe drug for testing in other models as it produces non-toxic effects in primary astrocytes. Combined, these effects suggest that SAC affords antioxidant properties and potential antitumor efficacy against GBM.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Uso da Toxina Botulínica Tipo A em pacientes com Neuralgia Trigeminal

This article provides a systematic review of the efficacy of Botulinum Toxin Type A (TBA) in treating trigeminal neuralgia (TN), a painful disorder affecting the trigeminal nerve. TN is described in the literature as a facial pain of acute nature, often unilateral, characterized by episodes of intense and transient pain, comparable to electric shocks. The diagnosis is made through a detailed evaluation that includes anamnesis, characterization of symptoms, and imaging tests to exclude similar pathologies. Conventional therapy for TN involves the use of carbamazepine; however, in cases of pharmacological resistance or ineffectiveness of standard treatments, TBA emerges as a viable therapeutic alternative. The efficacy of TBA, as demonstrated in retrospective and prospective studies, is due to its ability to inhibit the release of neurotransmitters responsible for pain transmission and inflammatory processes. Various studies corroborate the efficacy of TBA, especially in patients over 50 years old, observing substantial improvement in symptoms with minimal adverse effects. The administration of TBA is carried out directly in the trigger zones, where it has proven to be well-tolerated, providing significant improvement in the quality of life of the affected patients. Despite promising results, the literature suggests the need for additional studies to optimize the dosage protocols and application of TBA, aiming to ensure its efficacy and safety in the long term. In conclusion, this article reiterates that TBA represents an effective and minimally invasive therapeutic alternative for the treatment of TN. However, it is essential to develop individualized therapeutic protocols and conduct more comprehensive studies to consolidate its use as the treatment of choice.

Use of Essential Oils to Counteract the Phenomena of Antimicrobial Resistance in Livestock Species.

Antimicrobial resistance is an increasingly widespread phenomenon that is of particular concern because of the possible consequences in the years to come. The dynamics leading to the resistance of microbial strains are diverse, but certainly include the incorrect use of veterinary drugs both in terms of dosage and timing of administration. Moreover, the drug is often administered in the absence of a diagnosis. Many active ingredients in pharmaceutical formulations are, therefore, losing their efficacy. In this situation, it is imperative to seek alternative treatment solutions. Essential oils are mixtures of compounds with different pharmacological properties. They have been shown to possess the antibacterial, anti-parasitic, antiviral, and regulatory properties of numerous metabolic processes. The abundance of molecules they contain makes it difficult for treated microbial species to develop pharmacological resistance. Given their natural origin, they are environmentally friendly and show little or no toxicity to higher animals. There are several published studies on the use of essential oils as antimicrobials, but the present literature has not been adequately summarized in a manuscript. This review aims to shed light on the results achieved by the scientific community regarding the use of essential oils to treat the main agents of bacterial infection of veterinary interest in livestock. The Google Scholar, PubMed, SciELO, and SCOPUS databases were used for the search and selection of studies. The manuscript aims to lay the foundations for a new strategy of veterinary drug use that is more environmentally friendly and less prone to the emergence of drug resistance phenomena.

Age-Dependent Phenomena of 6-Hz Corneal Kindling Model in Mice.

Although numerous studies have acknowledged disparities in epilepsy-related disease processes between young and aged animals, little is known about how epilepsy changes from young adulthood to middle age. This study investigates the impact of aging on 6-Hz corneal kindling in young-adult mice and middle-aged mice. We found that the kindling acquisition of the 6-Hz corneal kindling model was delayed in middle-aged mice when compared to young-adult mice. While the seizure stage and incidence of generalized seizures (GS) were similar between the two age groups, the duration of GS in the kindled middle-aged mice was shorter than that in the kindled young-adult mice. Besides, all kindled mice, regardless of age, were resistant to phenytoin sodium (PHT), valproate sodium (VPA), and lamotrigine (LGT), whereas middle-aged mice exhibited higher levetiracetam (LEV) resistance compared to young-adult mice. Both age groups of kindled mice displayed hyperactivity and impaired memory, which are common behavioral characteristics associated with epilepsy. Furthermore, middle-aged mice displayed more pronounced astrogliosis in the hippocampus. Additionally, the expression of Brain-Derived Neurotrophic Factor (BDNF) was lower in middle-aged mice than in young-adult mice prior to kindling. These data demonstrate that both the acquisition and expression of 6-Hz corneal kindling are attenuated in middle-aged mice, while hippocampal astrogliosis and pharmacological resistance are more pronounced in this age group. These results underscore the importance of considering age-related factors when utilizing the 6-Hz corneal kindling model in mice of varying age groups.

A Retrospective Study on tDCS Treatment in Patients with Drug-Resistant Chronic Pain.

Background. Transcranial direct current stimulation (tDCS) of the primary motor cortex (M1) has an analgesic effect superior to a placebo in chronic pain. Some years ago, tDCS was implemented at the Hospital Nacional of Paraplegics (Toledo, Spain) to treat patients with pharmacological resistance to chronic pain. Objective. The main objectives of this study with tDCS were (1) to confirm the safety of one-year treatment; (2) to estimate the number of patients after one year in treatment; (3) to describe the effects of tDCS on the pain intensity during one-year treatment; and (4) to identify factors related to treatment success. Methods. This was a retrospective study conducted at the National Hospital for Paraplegics with 155 patients with pharmacologically resistant chronic pain. Anodal tDCS was applied over the M1 for 20 min at 1.5 mA for 10 treatment sessions from Monday to Friday (Induction phase), followed by 2-3 sessions per month (Maintenance phase). Pain intensity was assessed using a Visual Analogue Scale (VAS). Results. Anodal tDCS on M1 confirmed the reduction in the pain intensity. Moreover, 58% of outpatients completed one year of treatment. Only the VAS values obtained during the baseline influenced the response to treatment. Patients with a very high VAS at the baseline were more likely to not respond adequately to tDCS treatment. Conclusions. Anodal tDCS over M1 is an adequate therapy (safe and efficient) to treat drug-resistant chronic pain. Moreover, pain intensity at the start of treatment could be a predictor of patients' continuity with tDCS for at least one year.

Drug Response Profiling Informs Personalized Bridging to Cell Therapy for Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia

Introduction Treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL) remains challenging in spite of recent progress with immunotherapy and targeted small-molecule combinations. Growing awareness of the molecular complexity of the disease with subtype-specific cancer dependencies positions functional drug testing as a promising precision tool to improve the prediction of clinical drug efficacy and adapt treatment. We leverage automated, image-based Drug Response Profiling (DRP) and machine learning to deliver individualized therapeutic options in real time for ALL patients with urgent medical needs. Here we report on the DRP experience of 307 primary samples from 278 patients with BCP-ALL, T-ALL/T-LBL and mixed phenotypic acute leukemia (MPAL) registered from 2016 to 2022 in a non-interventional setting. Methods We have generated a reference set of 141 diagnostic samples from patients with high-risk (75%) or standard/medium-risk (25%) disease as defined by the clinical trial AIEOP-BFM ALL 2017, alongside 122 samples with first and 44 with more advanced relapse. We assayed cell viability at single-cell level from viably frozen bone marrow (BM) samples, co-cultured with hTERT-immortalized mesenchymal stromal cells (MSC) and following a 72h drug incubation with a library of up to 110 compounds. We quantified cell counts at five concentrations in duplicates by segmenting and classifying MSC, live and dead leukemia cells using a modular image processing pipeline deployed on cloud infrastructure. We longitudinally confirmed assay consistency with PDX models and monitored integrity of the stromal layer on a per-sample basis. In a subset of cases (15-20%), relevant stromal detachment or low leukemia survival led to samples not meeting all quality control standards. Drugs were ranked by the area under the dose response curve (AUC) and referenced to all previous samples. This patient-specific ex vivo drug fingerprint was reported to the treating physician with a typical turn-around time of less than two weeks. Results Dexamethasone sensitivities measured ex vivo in BM samples from pediatric front-line patients, who were treated on a one-week cytoreductive steroid pre-phase followed by a four-drug induction regimen, correlated significantly with clinical responses at d8 (PGR vs. PPR, p<0.001), d15 (flow MRD below 10%, p<0.001) and d33 (PCR MRD below E-2, p<0.001). By integrating dose-response profiles from all patients, we observed differential drug activities across cytogenetic groups and identified exceptional responders to Venetoclax in very high-risk subtypes including TCF3::HLF (5 patients, AUC percent rank: 0.03 - 0.19) as well as refractory T-ALL. In 77 r/r T-ALL patients from 12 different countries, DRP supported selection of a personalized bridging regimen prior to consolidation with cellular therapy. Top-scoring drug sensitivities (10th percentile of AUC) were found for a variety of agents including Imatinib, Dasatinib, Venetoclax, Bortezomib, Selinexor and different nucleoside analogs. Eleven T-ALL and 5 BCP-ALL patients received a DRP-guided bridging element involving compounds with high ex vivo drug efficacy and complemented by chemotherapy (Table 1). The other 66 r/r patients were treated according to physician's choice incorporating information on drug resistance detected by DRP in real time. Bridging strategies implementing DRP-guided therapy blocks stabilized or reduced disease burden in 15/16 patients allowing HSCT or infusion of CAR-T cells in 12 patients with 8 patients still being in remission at last follow-up. Conclusion We describe an array of pharmacological sensitivity and resistance patterns across a heterogeneous, high-risk cohort of BCP- and T-ALL patients. We identified individual susceptibilities to targeted agents of different classes including BH3-mimetics, proteasome, tyrosine kinase and exportin inhibitors as well as standard-of-care chemotherapeutics. Prospective recording of drug response profiles offers opportunities to systematically correlate ex vivo and clinical responses and inform personalized therapies. These data strongly advocate inclusion of DRP into randomized, early and late phase clinical trials for high-risk pediatric ALL, comprehensively integrating molecular and functional precision information to improve patient stratification and long-term outcome after HSCT and CAR-T.

Biogenic Silver Nanoparticles and Stressors Generate Synergistic Growth Inhibition in Candida Species through Cell Wall Damage, Osmotic Stress, and Oxidative Stress.

The need to combat and reduce the incidence, virulence, and drug resistance of species belonging to Candida genus, has led to the development of new strategies. Nanotechnology, through the implementation of nanomaterials, has emerged as an infallible tool to treat various diseases caused by pathogens, where its mechanisms of action prevent the development of undesirable pharmacological resistance. The antifungal activity and adjuvant properties of biogenic silver nanoparticles in different Candida species (C. parapsilosis, C. glabrata, and C. albicans) are evaluated. The biogenic metallic nanoparticles were developed by quercetin-mediated biological synthesis. The physicochemical properties were studied by light scattering, electrophoretic mobility, UV-vis and infrared spectroscopy, and transmission electron microscopy. The elucidation of mechanisms of antifungal action was carried out under stress conditions in Candida species at the cell wall and response to oxidative stress. Small silver nanoparticles (≈ 16.18 nm) with irregular morphology, and negative surface electrical charge (≈ -48.99 mV), were obtained through quercetin-mediated biosynthesis. Infrared spectra showed that the surface of silver nanoparticles is functionalized with the quercetin molecule. The antifungal activity of biogenic nanoparticles had efficacy in the following trend C. glabrata ≥ C. parapsilosis > C. albicans. Biogenic nanoparticles and stressors showed synergistic and potentiated antifungal effects through cell damage, osmotic stress, cell wall damage, and oxidative stress. Silver nanoparticles synthesized by quercetin-mediated biosynthesis could be implemented as a powerful adjuvant agent to enhance the inhibition effects of diverse compounds over different Candida species.

Ultrastructural alterations due to sodium metavanadate treatment in the blood stages of Plasmodium yoelii yoelii

Malaria is a potentially mortal disease caused by parasites of the genus Plasmodium spp. It has a wide distribution in the world and unfortunately there are several factors that make its control difficult; among which the development of pharmacological resistance to the different drugs used to treat this disease stands out, which makes it necessary to design new compounds that have an antimalarial effect. Previous studies have shown that vanadium has a broad antiparasitic spectrum and is also safe for the host, so the objective of this research was to evaluate the antimalarial potential of sodium metavanadate (SM) and to analyze the ultrastructural changes in parasites exposed. The method consisted of inoculating CD-1 male mice with Plasmodium yoelii yoelii and administering a 10 mg/kg/day dose of SM orally for 4 days. On the fifth day, whole blood samples were obtained, processed for ultrastructural analysis, and the changes in the different parasite stages were compared against the control. Our results showed that SM decreased parasitemia compared to the group that did not receive treatment and modified the ultrastructure in all parasitic stages because it damaged the membranes, causing alterations mainly in the nucleus and in the mitochondria as well as the loss of cellular organization, which could affect the integrity of these parasites and decrease its viability.

In Vitro Antifungal Activity of Three Synthetic Peptides against Candida auris and Other Candida Species of Medical Importance.

Candidiasis is an opportunistic infection affecting immunosuppressed and hospitalized patients, with mortality rates approaching 40% in Colombia. The growing pharmacological resistance of Candida species and the emergence of multidrug-resistant Candida auris are major public health problems. Therefore, different antimicrobial peptides (AMPs) are being investigated as therapeutic alternatives to control candidiasis effectively and safely. This work aimed to evaluate the in vitro antifungal activity of three synthetic AMPs, PNR20, PNR20-1, and 35409, against ATCC reference strains of Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida tropicalis, and clinical isolates of C. auris. Antifungal susceptibility testing, determined by broth microdilution, showed that the AMPs have antifungal activity against planktonic cells of all Candida species evaluated. In C. auris and C. albicans, the peptides had an effect on biofilm formation and cell viability, as determined by the XTT assay and flow cytometry, respectively. Also, morphological alterations in the membrane and at the intracellular level of these species were induced by the peptides, as observed by transmission electron microscopy. In vitro, the AMPs had no cytotoxicity against L929 murine fibroblasts. Our results showed that the evaluated AMPs are potential therapeutic alternatives against the most important Candida species in Colombia and the world.

Essential Oils for a Sustainable Control of Honeybee Varroosis.

The Varroa destructor parasite is the main obstacle to the survival of honey bee colonies. Pest control mainly involves the use of synthetic drugs which, used with the right criteria and in rotation, are able to ensure that infestation levels are kept below the damage threshold. Although these drugs are easy to use and quick to apply, they have numerous disadvantages. Their prolonged use has led to the emergence of pharmacological resistance in treated parasite populations; furthermore, the active ingredients and/or their metabolites accumulate in the beehive products with the possibility of risk for the end consumer. Moreover, the possibility of subacute and chronic toxicity phenomena for adult honeybees and their larval forms must be considered. In this scenario, eco-friendly products derived from plant species have aroused great interest over the years. In recent decades, several studies have been carried out on the acaricidal efficacy of plant essential oils (EOs). Despite the swarming of laboratory and field studies, however, few EO products have come onto the market. Laboratory studies have often yielded different results even for the same plant species. The reason for this discrepancy lies in the various study techniques employed as well as in the variability of the chemical compositions of plants. The purpose of this review is to take stock of the research on the use of EOs to control the V. destructor parasite. It begins with an extensive discussion of the characteristics, properties, and mechanisms of action of EOs, and then examines the laboratory and field tests carried out. Finally, an attempt is made to standardize the results and open up new lines of study in future.

Bifrontal electroconvulsive therapy leads to improvement of cerebral glucose hypometabolism in frontotemporal dementia with comorbid psychotic depression – a case report

BackgroundDifferentiating depression and dementia in elderly patients represents a major clinical challenge for psychiatrists. Pharmacological and non-pharmacological treatment options for both conditions are often used cautiously due to fear of adverse effects. If a clinically indicated therapy is not initiated due to fear of adverse effects, the quality of life of affected patients may significantly be reduced.Case presentationHere, we describe the case of a 65-year-old woman who presented to the department of psychiatry of a university hospital with depressed mood, pronounced anxiety, and nihilistic thoughts. While several pharmacological treatments remained without clinical response, further behavioral observation in conjunction with 18F-fluoro‐2‐deoxy‐D‐glucose positron emission tomography/computed tomography (18F-FDG PET/CT) revealed the diagnosis of frontotemporal dementia (FTD). To counter the pharmacological treatment resistance of psychotic depression, we decided to perform electroconvulsive therapy (ECT). Remarkably, ten sessions of ECT yielded an almost complete remission of depressive symptoms. In addition, the patient’s delusional ideas disappeared. A follow-up 18F-FDG PET/CT after the ECT series still showed a frontally and parieto-temporally accentuated hypometabolism, albeit with a clear regression compared to the previous image. The follow-up 18F-FDG PET/CT thus corroborated the diagnosis of FTD, while on the other hand it demonstrated the success of ECT.ConclusionsIn this case, ECT was a beneficial treatment option for depressive symptoms in FTD. Also, 18F-FDG PET/CT should be discussed as a valuable tool in differentiating depression and dementia and as an indicator of treatment response.

Whole-genome resequencing in the sea louse Caligus rogercresseyi uncovers gene duplications and copy number variants associated with pesticide resistance

The sea louse Caligus rogercresseyi is a marine ectoparasite that constitutes one of the major threats to the salmon farming industry, where the primary control strategy is the use of delousing drugs through immersion treatments. The emergence of pharmacological resistance in this copepodid species has previously been described using transcriptome data. However, the molecular mechanisms underlying chromosome rearrangements have not yet been explored. This study aimed to identify structural genomic variations and gene expression in C. rogercresseyi associated with pesticide sensitivity. In this study, genome resequencing was conducted using Oxford Nanopore Technology on lice strains with contrasting sensitivity to azamethiphos to detect genome duplications. Transcriptome profiling of putative gene duplications was performed by Illumina sequencing. Copy Number Variants (CNVs) were identified through comparative coverage, and collinear/tandem gene duplications over all the chromosomal regions by sequence homology. Duplications or CNVs in functional genes were primarily identified in transposable elements and genes related to the drug response, with differential expression values calculated by RNA-seq analyses of the same strains. Notably, differentially duplicated genes were found in coding regions related to cuticle proteins, suggesting that a putative resistance mechanism may be associated with cuticular structure formation and the proteins involved. Collectively, the results revealed that the intensive use of pesticides on sea lice populations increases the frequency of gene duplication, expanding the molecular elements involved in drug response. This study is the first to report an association between genome rearrangements and pharmacological resistance in sea lice populations.

Binding Analysis and Structure-Based Design of Tricyclic Coumarin-Derived MTHFD2 Inhibitors as Anticancer Agents: Insights from Computational Modeling.

Unfolded protein response (UPR)-dependent metabolic reprogramming diverts metabolites from glycolysis to mitochondrial 1C metabolism, highlighting pharmacological resistance to folate drugs and overexpression of certain enzymes. Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme that plays a key role in 1C metabolism in purine and thymidine synthesis and is exclusively overexpressed in cancer cells but absent in most healthy adult human tissues. To the best of our knowledge, tricyclic coumarin-based compounds (substrate site binders) and xanthine derivatives (allosteric site binders) are the only selective inhibitors of MTHFD2 reported until the present date. The current study aims at the investigation of the available structural data of MTHFD2 in complex with potent and selective inhibitors that occupy the substrate binding site, further providing insights into binding mode, key protein-ligand interactions, and conformational dynamics, that correspond to the experimental binding affinities and biological activities. In addition, we carried out structure-based drug design on the substrate binding site of MTHFD2, by exploiting the cocrystal structure of MTHFD2 with the tricyclic coumarin-based inhibitor. The structure-based drug design campaign involves R-group enumeration, bioisostere replacement, molecular docking, ADME prediction, MM-GBSA binding free energy calculations, and molecular dynamics simulations, that led to a small library of new and potential compounds, capable of selectively inhibiting MTHFD2. The results reported herein are expected to benefit medicinal chemists working on the development of selective MTHFD2 inhibitors for cancer treatment, although experimental validation by biochemical and/or pharmacokinetic assays is required to substantiate the outcomes of the study.

Abstract 1631: Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs)

Abstract GISTs are common malignant mesenchymal tumors that occur in the GI tract, with an estimated incidence rate of 1% to 2%. About 75% to 80% of patients with GIST have mutations in the KIT gene, while 5% to 10% have mutations in the platelet-derived growth factor receptor α (PDGFRA) gene. Tyrosine kinase inhibitors (TKIs) offer patients an improved quality of life but increased pharmacological resistance to TKIs is often observed. BCL-2 is expressed in >80% of GISTs, and amplification of BCL-2 and BCL-xL are common features associated with disease progression. One approach to enhance GIST eradication is to concurrently inhibit oncogenic KIT signaling while actively engaging apoptotic pathways. Olverembatinib (HQP1351) is a new, third-generation TKI that targets BCR-ABL1, KIT and PDGFRA and is currently in development for relapsed or refractory chronic myeloid leukemia and GIST. Lisaftoclax (APG-2575) is a selective BCL-2 inhibitor under development for hematologic malignancies. The purpose of this study was to evaluate whether combining a BCL-2 inhibitor, lisaftoclax, with olverembatinib enhances treatment effect on imatinib-resistant GISTs. Compared to imatinib-sensitive GIST cell lines (GIST882 and GIST-T1), increased BCL-2 protein expression was observed in imatinib-resistant GIST cells, particularly in GIST430, GIST48, and GIST48B lines. Cell-based antiproliferation studies showed additive activity of olverembatinib and lisaftoclax in imatinib-resistant GIST cells. In vivo studies using imatinib-resistant GIST430 cell-derived xenograft models further revealed that coadministration of olverembatinib at 15 mg/kg (every other day for 3 weeks) with lisaftoclax at 50 mg/kg (daily for 3 weeks) resulted in enhanced antitumor effects compared to either agent alone. Tumor growth inhibition (TGI) rates for combined treatment with olverembatinib and lisaftoclax reached 76.8%, which was superior to the TGI rate of either agent alone: 57.6% and 31.2% for olverembatinib and lisaftoclax, respectively. Regarding putative mechanisms, olverembatinib treatment was shown to downregulate BCL-xL and MCL-1 protein expression, possibly via inhibition of KIT signal pathway and downstream signal transducer and activator of transcription 3 (STAT3). Lisaftoclax triggered apoptosis by disrupting the BCL-2:BIM complex. Therefore, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly augmented cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1), thus triggering apoptosis and subsequently enhancing antitumor effects. Our results demonstrate that olverembatinib and BCL-2 inhibitor lisaftoclax have additive antitumor effects in imatinib-resistant GIST. This novel dual approach may have the potential for treating patients with GISTs whose disease has progressed after treatment with TKIs. Citation Format: Ping Min, Guangfeng Wang, Eric Liang, Bingxing Wu, Huidan Yu, Dajun Yang, Yifan Zhai. Combination of olverembatinib (HQP1351) with BCL-2 inhibitor lisaftoclax (APG-2575) overcomes resistance in gastrointestinal stromal tumors (GISTs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1631.

Abstract P2-26-02: Valproic acid induces apoptosis and mitochondrial perturbations in breast cancer cells

Abstract Introduction: Breast cancer is the second leading cause of death among women after lung cancer. Despite the increase and improvement in preventive screening, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the rapid development of resistance to drug treatments, which recalls the complicity of multiple factors. Breast cancer prognosis can be correlated with specific histological subtypes and their receptor status. Depending on stage and invasiveness, ER + and PR + breast cancers are treated with surgery and adjuvant chemotherapy, which includes endocrine-based agents, such as SERMS (Tamoxifen), SERDs (Fulvestrant) and aromatase inhibitors (Letrazole and Anastrazole). Resistance to endocrine therapy can be explained by various mechanisms, such as mutations of the estrogen receptor, epigenetic modifications of the same receptor, alterations in hormone synthesis and metabolism, aberrant activation of signaling transduction pathways (up-regulation of PI3K, MAPK, CDKs). To reduce pharmacological resistance, it is possible to use histone deacetylase inhibitors, such as valproic acid, as the epigenetic alterations play an important role in the oncogenesis of breast cancer. Epigenetic modifications of genes are a mechanism used by cancer cells to silence the expression of tumor suppressor genes, driven by epigenetic mutations of regulator factors. The valproic acid, used as an antiepileptic drug and for the treatment of bipolar disorders, is also a selective inhibitor of class I and class II HDAC isoforms, involved in the progression of different types of tumors. In this study, we tested the effects of valproic acid on the signaling involved in apoptosis and in the generation of Reactive Oxygen Species (ROS) in MCF-7 breast cancer cells. Methods: MTT assay was performed to evaluate cell proliferation. Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, while protein levels were detected by Western Blotting (WB). Results: Cell treatment with valproic acid reduced cell proliferation and induced G0/G1 cell cycle arrest. In addition, the drug enhanced the generation of ROS by the mitochondria and addressed breast cell death by apoptosis. Indeed, the valproic acid was found to downregulate the anti-apoptotic marker Bcl-2 and to upregulate the expression of pro-apoptotic markers, such as Bax and Bad, leading to release of cytochrome C into the cytosol and PARP cleavage. The drastic reduction in mitochondrial membrane potential and in mitochondrial mass was observed following treatment with valproic acid, indeed the ratio between the two parameters, index of functionality per mitochondrion, resulted to be lowered, thus emphasizing the metabolic perturbation in the cells. Conclusion: In summary, our results have demonstrated that in estrogen receptor positive breast cancer cells the valproic acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. This study encourages that valproic acid, as an epigenetic drug, is a promising class of antineoplastic agent to be further explored also in combination with other classical chemotherapeutics in breast tumors. Citation Format: Alessandro Paolì, Martina Forestiero, Giuseppina Daniela Naimo, Loredana Mauro, Maria Luisa Panno, Francesca Giordano. Valproic acid induces apoptosis and mitochondrial perturbations in breast cancer cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-02.

The prevalence of drug resistance to antiretroviral therapy in the Republic of Mordovia

Objective. To study the causes of virological inefficiency of ART and to analyze the prevalence of mutations of resistance to ART drugs among HIV-infected patients, who lives in the Republic of Mordovia and receiving ART.Materials and methods. We studied 37 blood tests with detectable viral load from HIV-infected patients receiving observation in the GBUZ RM «MRTSPBSPID», taking ART for more than 24 weeks. We The evaluated patient’s adherence to treatment. The analysis for drug resistance was performed by genotyping, using HIV-Resist-Seg diagnostic test systems, (the Federal State Budgetary Institution of the Central Research Institute of Rospotrebnadzor, Russia). Statistical data processing (c2 calculation) StatSoft, Inc. (2011), STATISTICA, Russia.Results and discussion: in the group of patients with mutations of resistance to antiretroviral drugs, mutations of pharmacological resistance to drugs of two and three groups are most often determined (in 87.5% of cases). The number and frequency of genetic aberrations in general did not depend on the number of already approved treatment regimens. At the same time, in patients with two or more changes of ART regimens in the anamnesis, the occurrence of mutations of drug resistance to IP was significantly higher than in HIV-infected patients with one ART regimen.Conclusion: in the Republic of Mordovia, virological inefficiency is associated equally with both a violation of ART intake (poor adherence) and the development of pharmacoresistance. It is important to determine the drug resistance of HIV in patients receiving ART.

Role of filamin A in the pathogenesis of neuroendocrine tumors and adrenal cancer.

Cell cytoskeleton proteins are involved in tumor pathogenesis, progression and pharmacological resistance. Filamin A (FLNA) is a large actin-binding protein with both structural and scaffold functions implicated in a variety of cellular processes, including migration, cell adhesion, differentiation, proliferation and transcription. The role of FLNA in cancers has been studied in multiple types of tumors. FLNA plays a dual role in tumors, depending on its subcellular localization, post-translational modification (as phosphorylation at Ser2125) and interaction with binding partners. This review summarizes the experimental evidence showing the critical involvement of FLNA in the complex biology of endocrine tumors. Particularly, the role of FLNA in regulating expression and signaling of the main pharmacological targets in pituitary neuroendocrine tumors, pancreatic neuroendocrine tumors, pulmonary neuroendocrine tumors and adrenocortical carcinomas, with implications on responsiveness to currently used drugs in the treatment of these tumors, will be discussed.