Low Potency Research Articles

Covalent Inhibition of a Host-Pathogen Protein-Protein Interaction Reduces the Infectivity of Streptococcus pneumoniae.

The ever-expanding antibiotic resistance urgently calls for novel antibacterial therapeutics, especially those with a new mode of action. We report herein our exploration of protein-protein interaction (PPI) inhibition as a new mechanism to thwart bacterial pathogenesis. Specifically, we describe potent and specific inhibitors of the pneumococcal surface protein PspC, an important virulence factor that facilitates the infection of Streptococcus pneumoniae. Specifically, PspC has been documented to recruit human complement factor H (hFH) to suppress host complement activation and/or promote the bacterial attachment to host tissues. The CCP9 domain of hFH was recombinantly expressed to inhibit the PspC-hFH interaction as demonstrated on live pneumococcal cells. The inhibitor allowed for the first pharmacological intervention of the PspC-hFH interaction. This PPI inhibition reduced pneumococci's attachment to epithelial cells and also resensitized the D39 strain of S. pneumoniae for opsonization. Importantly, we have further devised covalent versions of CCP9, which afforded long-lasting PspC inhibition with low nanomolar potency. Overall, our results showcase the promise of PPI inhibition for combating bacterial infections as well as the power of covalent inhibitors.

In vitro activation of the CB1 receptor by the semi-synthetic cannabinoids hexahydrocannabinol (HHC), hexahydrocannabinol acetate (HHC-O) and hexahydrocannabiphorol (HHC-P).

Semi-synthetic cannabinoids (SSCs) including hexahydrocannabinol (HHC) are emerging on the drug market and sold openly as purportedly legal replacements for cannabis and Δ9-THC. By the beginning of 2024, 24 European countries had identified HHC, often sold openly in edibles (foods/candy), vapes and low-THC cannabis flowers and resins. The SSC market is developing rapidly, with HHC acetate (HHC-O), hexahydrocannabiphorol (HHC-P) and others recently identified. These developments may mark the first major change in the market for 'legal' replacements to cannabis since 'Spice' containing synthetic cannabinoids, such as JWH-018, emerged in 2008. Currently, there are some data available on the pharmacology of SSCs, which is crucial for understanding their effects, evaluating health risks and informing public health responses. This study focused on characterizing the in vitro activation of the human CB1 receptor by the (R)- and (S)-epimers of HHC, HHC-P and HHC-O. Using recombinant CHO-K1 cells expressing the human CB1 receptor, the potency (EC50) and efficacy were determined. It was established that (9R)-HHC and (9R)-HHC-P activated the CB1 receptor as partial agonists and with five and two times lower potency compared to JWH-018, respectively, while the (S)-epimers exhibited even lower potency. The (R)-epimer of HHC-O activate the CB1 receptor to even lesser extent and the (S)-epimer showed no activation. For HHC and HHC-P, all epimers exhibited similar level of efficacy. This available evidence suggests cannabimimetic effects of the tested SSC with the exception for the acetates that likely function as pro-drugs in vivo.

Potent, Selective Agonists for the Cannabinoid-like Orphan GProtein-Coupled Receptor GPR18: A Promising Drug Target forCancer and Immunity.

The human orphan G protein-coupled receptor GPR18, activated by Δ9-tetrahydrocannabinol (THC), constitutes a promising drug target in immunology and cancer. However, studies on GPR18 are hampered by the lack of suitable tool compounds. In the present study, potent and selective GPR18 agonists were developed showing low nanomolar potency at human and mouse GPR18, determined in β-arrestin recruitment assays. Structure-activity relationships were analyzed, and selectivity versus cannabinoid (CB) and CB-like receptors was assessed. Compound 51 (PSB-KK1415, EC50 19.1 nM) was the most potent GPR18 agonist showing at least 25-fold selectivity versus CB receptors. The most selective GPR18 agonist 50 (PSB-KK1445, EC50 45.4 nM) displayed >200-fold selectivity versus both CB receptor subtypes, GPR55, and GPR183. The new GPR18 agonists showed minimal species differences, while THC acted as a weak partial agonist at the mouse receptor. The newly discovered compounds represent the most potent and selective GPR18 agonists reported to date.

Sensitivity Differences in Testing of Polyurethane Condoms’ Lubricant Compatibility

Abstract Over-the-counter (OTC) personal lubricants (PLs) generally have less potent effects on condoms’ mechanical properties than do appropriate positive controls. Therefore, to assess the compatibility of OTC PLs with condoms, a test method must be more sensitive than those employed to capture the effect of a strong positive control. Using various percent compositions of selected common ingredients in commercial PLs, we attempted to differentiate between the sensitivities of different methods for testing lubricant effects on polyurethane (PU) condoms. After discrete applications of each composition, we studied induced stresses and stress-to-strain ratios at various strain rates, and stress reduction at a constant stretch, in PU condom coupons at 37°C. Swelling was studied at room temperature (RmT). Changes in the induced stress and stress-to-strain ratio because of low percent composition of a potent surrogate component were detectable only at low strain rates. A few low percent compositions of potent ingredients – though they induced insignificant changes in the material as detected by tensile testing and did not swell the PU material – noticeably reduced the percent stress at a constant stretch. Slower strain rates in tensile testing were necessary to detect the effects of low potency combinations, whereas stress reduction was the most efficient detector of change at a constant physiologically relevant strain. Absence of swelling and insignificant changes in tensile properties at high strain rates did not guarantee an absence of interactions. Manufacturers now have a more sensitive method for capturing the interactions of PU condoms with OTC PLs, which are generally less potent than appropriate positive controls.

In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on μ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (β-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

Evaluation of methyl salicylate, limonene, and thymol as potential biocidal against Trogoderma granarium everts larvae

The investigation of plant-based natural products, especially essential oils, as insecticide alternatives, responds to challenges as induced resistance and environmental drawbacks linked to prolonged synthetic insecticide use. Although essential oils demonstrate promising activity, their utilization faces hurdles related to standardization. In this study, contact/fumigation properties of three molecules (methyl salicylate, limonene and thymol) known for their activity against stored product beetles, were tested in laboratory bioassays toward young and old larval stages of Trogoderma granarium. The experiment revealed significant differences in their effects based on larval age and chemical concentration. Methyl salicylate exhibited concentration-dependent effects on both young and old larvae, with higher concentrations leading to accelerated mortality rates. For instance, young larvae exposed to 10 mg displayed an LT50 of 33.75 h, while old larvae showed an LT50 of 39.07 h. Similarly, at a concentration of 5 mg, young and old larvae exhibited LT50 values of 72.79 and 79.68 h, respectively, after treatment. Limonene also exhibited concentration-dependent effects, although with lower potency compared to methyl salicylate. Thymol showed efficacy only against young larvae of T. granarium. Statistical analyses confirmed the significant impact of each chemical on larval survival time, with tested chemicals displaying significant concentration-dependent variations in mortality rates. These findings suggest the potential of methyl salicylate, and to a lesser extent limonene and thymol, as effective biocidal agents against T. granarium larvae.

An in Vitro triple screen model for human mineralocorticoid receptor activity

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18β-glycyrrhetinic acid (18β-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Antigenic operon fragmentation and diversification mechanism in Bacteroidota impacts gut metagenomics and pathobionts in Crohn's disease microlesions

ABSTRACT Comensal Bacteroidota (Bacteroidota) and Enterobacteriacea are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism in Bacteroidota remain unclear. By using the classical lipopolysaccharide/O-antigen ‘rfb operon’ in Enterobacteriaceae as a surface antigen model (5-rfb-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification, we characterized the integrity and conservancy of the entire rfb operon in Bacteroidota. Through exploratory analysis of complete genomes and metagenomes, we discovered that most Bacteroidota have the rfb operon fragmented into nonrandom patterns of gene-singlets and doublets/triplets, termed ‘rfb-gene-clusters’, or rfb-‘minioperons’ if predicted as transcriptional. To reflect global operon integrity, contiguity, duplication, and fragmentation principles, we propose a six-category (infra/supra-numerary) cataloging system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in gut-wall specific micro-niches or micropathologies. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes. DNA insertions, overrepresenting DNA-exchange-avid (Bacteroides) species, impact our interpretation of functional metagenomics data by inflating by inflating gene-based pathway inference and by overestimating ‘extra-species’ abundance. Of disease relevance, Bacteroidota species isolated from cavitating/cavernous fistulous tract (CavFT) microlesions in Crohn’s Disease have supra-numerary fragmented operons, stimulate TNF-alpha from macrophages with low potency, and do not induce hyperacute peritonitis in mice compared to CavFT Enterobacteriaceae. The impact of ‘foreign-DNA’ insertions on pro-inflammatory operons, metagenomics, and commensalism/opportunism requires further studies to elucidate their potential for novel diagnostics and therapeutics, and to elucidate the role of co-existing pathobionts in Crohn’s disease microlesions.

A dose titration protocol for once-daily insulin glargine 300 U/mL for the treatment of diabetes mellitus in dogs.

In purpose-bred dogs, insulin glargine 300 U/mL (IGla300) has long duration of action, peakless time-action profile, and low potency, making it suitable for use as a basal insulin. To evaluate IGla300 in client-owned diabetic dogs monitored using a flash glucose monitoring system (FGMS). Ninety-five client-owned diabetic dogs, newly diagnosed or previously treated with other insulin formulations, with or without concurrent diseases. Prospective multi-institutional study. Clinical signs and standardized assessment of FGMS data, using treatment and monitoring guidelines established a priori, guided dose adjustments and categorization into levels of glycemic control. The initial IGla300 dose was 0.5 U/Kg q24h for newly diagnosed dogs and (median dose [range]) 0.8 U/Kg (0.2-2.5) q24h for all dogs. Glycemic control was classified as good or excellent in 87/95 (92%) dogs. The IGla300 was administered q24h (1.9 U/kg [0.2-5.2]) and q12h (1.9 U/kg/day [0.6-5.0]) in 56/95 (59%) and 39/95 (41%) dogs, respectively. Meal-time bolus injections were added in 5 dogs (0.5 U/kg/injection [0.3-1.0]). Clinical hypoglycemia occurred in 6/95 (6%) dogs. Dogs without concurrent diseases were more likely to receive IGla300 q24h than dogs with concurrent diseases (72% vs 50%, respectively; P = .04). Insulin glargine 300 U/mL can be considered a suitable therapeutic option for once-daily administration in diabetic dogs. Clinicians should be aware of the low potency and wide dose range of IGla300. In some dogs, twice-daily administration with or without meal-time bolus injections may be necessary to achieve glycemic control. Monitoring with FGMS is essential for dose titration of IGla300.

Pharmacokinetics, Tolerability, and Safety of Esmethadone in Subjects with Chronic Kidney Disease or Hepatic Impairment.

Esmethadone (dextromethadone; d-methadone; S-methadone (+)-methadone; REL-1017) is a low potency N-methyl-D-aspartate (NMDA) receptor channel blocker that showed a rapid and sustained adjunctive antidepressant effects in patients with major depressive disorder with inadequate response to ongoing serotonergic antidepressant treatment. Previous studies indicated that esmethadone is partially excreted by the kidney (53.9% of the dose) and by the liver (39.1% of the dose). Here we studied the pharmacokinetics and safety of esmethadone after a single oral dose of 25 mg in subjects with different stages of kidney and liver impairment. In subjects with a mild and moderate decrease in glomerular fraction rate (GFR), esmethadone Cmax and AUC0-inf values did not differ compared with healthy subjects. In patients with severe renal impairment, the ratios of Cmax and AUC0-inf values compared with healthy subjects were above 100% (138.22-176.85%) and, while modest, these increases reached statistical significance. In subjects with end stage renal disease (ESRD) undergoing intermittent hemodialysis (IHD), Cmax and AUC0-inf values were not statistically different compared with healthy subjects. IHD did not modified plasma total esmethadone concentrations in blood exiting versus entering the dialyzer. Dose adjustment is not warranted in subjects with mild-to-moderate impaired renal function. Dose reduction may be considered for select patients with severe renal disfunction. In subjects with mild-or-moderate hepatic impairment, Cmax and AUC0-inf were approximately 20-30% lower compared with healthy controls. The drug free fraction increased with the severity of hepatic impairment, from 5.4% in healthy controls to 8.3% in subjects with moderate hepatic impairment. Mild and moderate hepatic impairment has a minimal to modest impact on exposure to total or unbound esmethadone and dose adjustments are not warranted in subjects with mild and moderate hepatic impairment. Administration of esmethadone was well tolerated in healthy adult subjects, in subjects with mild or moderate hepatic impairment, and in subjects with mild moderate or severe renal impairment, including patients with ESRF undergoing dialysis.

A first-in-human, phase 1 study evaluating oral TACC3 inhibitor, AO-252, in advanced solid tumors.

TPS3176 Background: TACC3 is the most oncogenic member of the transforming acidic coiled-coil domain-containing protein (TACC) family, which have important roles in microtubule and centrosome-related processes. TACC3 is overexpressed in multiple cancers with centrosome amplification caused by genomic aberrations such as TP53 mutations, leading to chromosome instability and worse prognosis. AO-252 is a computationally designed, small molecule inhibitor of TACC3 protein-protein interactions. Binding assays and kinome screening suggested the specificity of AO-252 to TACC3. AO-252 targets the interactions of TACC3 with Clathrin/KIFC1, BRDA1 and MBD2/HAT complexes regulating mitosis, DNA damage response and epigenetic functions and was confirmed using co-immunoprecipitation (co-IP) assays. Gene expression analysis in multiple cell lines further confirmed the various TACC3 pathways affected by AO-252. In vitro, AO-252 showed low nanomolar potency in >200 cell line panel covering multiple different cancer indications and spared the normal cells. In vivo, AO-252 demonstrated strong tumor growth inhibition or regression in multiple xenograft models including triple-negative breast (TNBC), high-grade serous ovarian (HGSOC) and endometrial cancer with TP53 mutation/loss with a good therapeutic index. Methods: This first-in-human, multicenter, open-label phase I dose escalation and expansion study evaluates the safety, tolerability, maximum tolerated dose, and recommended phase 2 dose (RP2D) of AO-252 in solid tumors (NCT06136884). Patients with unresectable or metastatic TNBC, platinum-resistant HGSOC, primary peritoneal, fallopian-tube and serous endometrial cancers with documented TP53 mutation/loss, who have failed on at least 1 prior line of systemic therapy in advanced/metastatic setting (maximum 4 for TNBC and endometrial carcinoma) are eligible for enrollment. Part 1 follows an accelerated titration dose-escalation for cohort 1 and 2 and a traditional 3+3 study design from cohort 3. A maximum of 54 patients will be treated in the dose escalation, patients receiving AO-252 orally once or twice daily in a 28-day cycle. Part 2 consists of dose-expansion of a maximum of 30 patients into 2 RP2D levels of AO-252. Primary objectives are to evaluate the safety and tolerability as noted by dose-limiting toxicities, adverse events, serious adverse events, laboratory test results, electrocardiograms, vital signs, physical exams, and ECOG performance status. Secondary objectives are to determine the antitumor activity assessed by objective response rate per RECIST1.1, duration of response and disease control rate and to determine the pharmacokinetics of single and repeated doses of AO-252. Potential biomarkers relating to treatment outcome will be evaluated as exploratory endpoints. Enrollment in Cohort 1 is completed and enrollment to Cohort 2 will begin in February 2024. Clinical trial information: NCT06136884 .

Novel specific TCRs targeting KRASG12V-related cancers.

e14528 Background: KRAS mutations act as oncogenic drivers in a variety of tumors, such as pancreatic, colorectal and lung cancer. Among these, the KRASG12V mutation, being one of the most common mutations, is associated with adverse outcomes in patients. KRAS has been regarded as an “undruggable” target, and no clinical trials targeting tumors carrying shared KRASG12V mutation have been initiated. Notably, KRASG12D specific TCR-T has shown high effectiveness, including the regression of metastases in patients with pancreatic cancer. Therefore, TCR-engineered T cells could be a promising strategy for the treatment of solid tumors harboring G12V mutation. Methods: We have developed an innovative approach to rapidly and sensitively identify natural TCRs from single T cell derived from patients using CorreGene’s Single T cell TCR Cloning platform, thus obviating the requirement for NGS sequencing and TCR gene synthesis. Additionally, natural TCRs can be refined with SMART-TCR system, utilizing T cell reporter cell lines to construct a high-throughput T-cell Display Platform for functional screening of TCRs. This system is meticulously designed to enrich TCRs with superior potency and specificity from a natural TCR mutation library. We performed a thorough assessment of each TCR identified, evaluating them across both in vitro and in vivo settings. Results: In the present study, we identified three natural TCRs from patients' TILs, designated as KVA11-N01, KVA11-N02 and KVA11-N04 respectively, that specifically recognized the HLA-A*11:01-restricted KRAS G12V epitope. KVA11-N01 exhibited lower affinity and potency, prompting us to develop an affinity-optimized variant, KVA11-M01-231, using the SMART-TCR system. For comparison, we used the murine TCR TRAV3-301/BV401, identified by NCI from HLA-A11:01 transgenic mice immunized with KRAS G12V7-16, as a benchmark. Three of our TCRs, including KVA11-N02, KVA11-N04 and the engineered KVA11-M01-231, outperformed TRAV3-301/BV401 in both in vitro and in vivo assays. These three TCRs exhibited excellent expression intensity and stability on T cells, and notably, their IFN-γ release potency surpassed that of TRAV3-3*01/BV4*01. Furthermore, in vivo experiments confirmed the significant tumor suppression efficacy of our TCRs in a tumor-bearing mouse model, showing enhanced tumor growth inhibition compared to TRAV3-301/BV401. TCRs from Corregene’s platform exhibited exceptional specificity, with no allo-reactivity, no reaction to KRASG12V-negative cell lines, and no off-target effects. Additionally, all our TCRs demonstrates favorable tolerance in mice during in vivo studies. Conclusions: TCRs via Corregene’s Single T cell TCR Cloning platform and SMART-TCR system showed significantly excellent anti-tumor activities with great specificity and offers a promising solution for the treatment of KRASG12V-related cancers.

Migration of endocrine and metabolism disrupting chemicals from plastic food packaging

Plastics constitute a vast array of substances, with over 16000 known plastic chemicals, including intentionally and non-intentionally added substances. Thousands of chemicals, including toxic ones, are extractable from plastics, however, the extent to which these compounds migrate from everyday products into food or water remains poorly understood. This study aims to characterize the endocrine and metabolism disrupting activity, as well as the chemical composition of migrates from plastic food contact articles (FCAs) from four countries as significant sources of human exposure. Fourteen plastic FCAs covering seven polymer types with high global market shares were migrated into water and a water–ethanol mixture as food simulants according to European regulations. The migrates were analyzed using reporter gene assays for nuclear receptors relevant to human health and non-target chemical analysis to characterize the chemical composition. Chemicals migrating from each FCA interfered with at least two nuclear receptors, predominantly targeting pregnane X receptor (24/28 migrates). Moreover, peroxisome proliferator receptor gamma was activated by 19 out of 28 migrates, though mostly with lower potencies. Estrogenic and antiandrogenic activity was detected in eight and seven migrates, respectively. Fewer chemicals and less toxicity migrated into water compared to the water–ethanol mixture. However, 73 % of the 15 430 extractable chemical features also transferred into food simulants, and the water–ethanol migrates exhibited a similar toxicity prevalence compared to methanol extracts. The chemical complexity differed largely between FCAs, with 8 to 10631 chemical features migrating into food simulants. Using stepwise partial least squares regressions, we successfully narrowed down the list of potential active chemicals, identified known endocrine disrupting chemicals, such as triphenyl phosphate, and prioritized chemical features for further identification. This study demonstrates the migration of endocrine and metabolism disrupting chemicals from plastic FCAs into food simulants, rendering a migration of these compounds into food and beverages probable.

How to Change the Potency of a Disease Nosode Changes the Course of the Disease Itself

The work identified “active” and “inactive” chromosomes and how “inactive” chromosomes become “active”. For this purpose, a device for resonance diagnostics and therapy was used, connected to a computer, and the resonance of creation was used to transform inactive chromosomes into active ones. For this purpose, we added the sum of potencies to the existing chromosome values. So, for example, at the beginning of the study, the patient was tested with a low potency of chromosomes equal to cassette No. 3 and ended with testing of cassette No. 10. Next, we “added” and connected the potency found in cassette No. 32 with high potencies. As a result, testing now began with cassette No. 1, and ended with cassette No. 12, i.e. testing increased to 4 cassettes. In a 50-year-old man, in the initial state, testing of chromosome potency began with cassette No. 18, and ended with cassette No. 31, i.e. 13 cassettes were tested. After connecting a cassette with a sufficiently high potency (cassette No. 47), the start of testing changed and took place on cassette No. 2, and ended on cassette No. 49. Cassette testing increased to 49 cassettes, i.e. more than 3 times. A connection has been established between “inactive” chromosomes and various pathologies - the lower the chromosome activity, the higher the incidence. To cure diseases, we added the sum of potencies to the already existing chromosome potency values. As a result of transformations of chromosomes, as a result of their potentiation (increase in potency) when connecting a large cassette, the patient develops a new quality in the treatment of various diseases - those diseases begin to be treated that before the transformation occurred, before potentiation of chromosomes, treatment was not possible. Is it possible to potentiate not only chromosomes, but also other structures of the body? It turned out it was possible. Potentiation is possible not only with chromosomes, but also with disease nosodes. An 84-year-old patient had untreated calculous cholecystitis from which he suffered. However, after transforming the nosode, after potentiating the nosode, connecting large cassettes to the nosode “calculous cholecystitis”, the patient’s calculous cholecystitis was extremely effectively cured. Treatment of an 82-year-old patient with cribriform prostate cancer. 10 years ago, the patient underwent surgery to remove cribriform prostate cancer - radical prostatectomy. After 10 years, a relapse occurred, which was treated with radiotherapy. Our patient underwent 36 procedures, 5 procedures per week. At the end of the course of treatment, testing of cribriform prostate cancer showed that the cancer was still being tested, i.e. treatment has not been completed. We treated this patient using resonance therapy with changing, increasing potency of the nosode “cribrous prostate cancer.” We increased the potency of the nosode in the patient to the point where the cancer could no longer be tested, i.e. We have completed treatment for cribriform prostate cancer. Thus, as a result of transformations of chromosomes, as a result of their potentiation (increase in potency) when connecting a large cassette, the patient develops a new, higher quality of chromosomes. The effectiveness of the treatment of various diseases was achieved by us by increasing the potency of the nosode of the disease from which the patient suffered and using this potency for treatment.

Curcuminoid PBPD induces cuproptosis and endoplasmic reticulum stress in cervical cancer via the Notch1/RBP-J/NRF2/FDX1 pathway.

Curcumin has been shown to have antitumor properties, but its low potency and bioavailability has limited its clinical application. We designed a novel curcuminoid, [1-propyl-3,5-bis(2-bromobenzylidene)-4-piperidinone] (PBPD), which has higher antitumor strength and improves bioavailability. Cell counting kit-8 was used to detect cell activity. Transwell assay was used to detect cell invasion and migration ability. Western blot and quantitative polymerase chain reaction were used to detect protein levels and their messenger RNA expression. Immunofluorescence was used to detect the protein location. PBPD significantly inhibited the proliferation of cervical cancer cells, with an IC50 value of 4.16 μM for Hela cells and 3.78 μM for SiHa cells, leading to the induction of cuproptosis. Transcriptome sequencing analysis revealed that PBPD significantly inhibited the Notch1/Recombination Signal Binding Protein for Immunoglobulin kappa J Region (RBP-J) and nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathways while upregulating ferredoxin 1 (FDX1) expression. Knockdown of Notch1 or RBP-J significantly inhibited NRF2 expression and upregulated FDX1 expression, leading to the inhibition of nicotinamide adenine dinucleotide phosphate activity and the induction of oxidative stress, which in turn activated endoplasmic reticulum stress and induced cell death. The overexpression of Notch1 or RBP-J resulted in the enrichment of RBP-J within the NRF2 promoter region, thereby stimulating NRF2 transcription. NRF2 knockdown resulted in increase in FDX1 expression, leading to cuproptosis. In addition, PBPD inhibited the acidification of tumor niche and reduced cell metabolism to inhibit cervical cancer cell invasion and migration. In conclusion, PBPD significantly inhibits the proliferation, invasion, and migration of cervical cancer cells and may be a novel potential drug candidate for treatment of cervical cancer.

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.

Subcutaneous administration of a novel TRPM8 antagonist reverses cold hypersensitivity while attenuating the drop in core body temperature.

We extend the characterization of the TRPM8 antagonist VBJ103 with tests of selectivity, specificity and distribution, therapeutic efficacy of systemic administration against oxaliplatin-induced cold hyperalgesia and the impact of systemic administration on core body temperature (CBT). Selectivity at human TRPA1 and TRPV1 as well as in vitro safety profiling was determined. Effects of systemic administration of VBJ103 were evaluated in a model of oxaliplatin-induced cold hyperalgesia. Both peripheral and centrally mediated effects of VBJ103 on CBT were assessed with radiotelemetry. VBJ103 had no antagonist activity at TRPV1 and TRPA1, but low potency TRPA1 activation. The only safety liability detected was partial inhibition of the dopamine transporter (DAT). VBJ103 delivered subcutaneously dose-dependently attenuated cold hypersensitivity in oxaliplatin-treated mice at 3, 10 and 30 mg·kg-1 (n = 7, P < 0.05). VBJ103 (30 mg·kg-1) antinociception was influenced by neither the TRPA1 antagonist HC-030031 nor the DAT antagonist GBR12909. Subcutaneous administration of VBJ103 (3, 10 and 30 mg·kg-1, but not 100 or 300 mg·kg-1, n = 7) decreased CBT (2°C). Intraperitoneal (i.p.) administration of VBJ103 (3, 10 and 30 mg·kg-1) dose-dependently decreased CBT to an extent larger than that detected with subcutaneous administration. Intracerebroventricular (i.c.v.) administration (306 nmol/1 μL; n = 5) did not alter CBT. We achieve therapeutic efficacy with subcutaneous administration of a novel TRPM8 antagonist that attenuates deleterious influences on CBT, a side effect that has largely prevented the translation of TRPM8 as a target.

#2188 Challenges in lipid control in the advanced chronic kidney disease clinic: findings from a cross-sectional study

Abstract Background and Aims Chronic kidney disease (CKD) is associated with lipid metabolism disruptions and heightens cardiovascular (CV) risk. International guidelines categorize CKD patients into high CV risk (LDL target &amp;lt; 70 mg/dL and a reduction &amp;gt;50% from baseline) for CKD G3b A1, CKD G3a A2, and CKD G2 A3; and very high CV risk (LDL target &amp;lt; 55 mg/dL and a reduction &amp;gt;50% from baseline) for CKD G4 and G5, CKD G3b A2-3, and those with type 2 diabetes and G3a A2-3, G3b regardless of UACR, or UACR &amp;gt; 300 mg/g regardless of eGFR. This cross-sectional study aims to evaluate the challenges of achieving LDL targets in advanced CKD patients, focusing on statin use. Method A cross-sectional study enrolled 312 patients with advanced CKD. Collected data included demographics, renal function stage, albuminuria/proteinuria, diabetes, and cardiovascular history. Documented statin use specified type and potency. Statistical analysis employed descriptive measures. Results Of the 312 patients, 207 were males, with a mean age of 69.18 ± 10.26 years, mean eGFR of 27.54 ± 12.22, and LDL of 86.64 ± 32.9. The majority had a history of diabetes (158) and previous cardiovascular disease (100). 69.6% used statins, with atorvastatin being the most common (165), followed by simvastatin (26), pravastatin (11), rosuvastatin (10), and pitavastatin (5). Statin potency varied, with high potency in 49.4%, moderate in 49.2%, and low in 1.4%. Only 19.6% of patients achieved adequate lipid control per guidelines. Conclusion This study underscores the high prevalence of dyslipidemia and the challenges in achieving LDL targets in advanced CKD patients. Despite the presence of cardiovascular risk factors, including a history of cardiovascular events, the majority did not attain adequate lipid control, highlighting the imperative to address therapeutic inertia to prevent LDL detrimental consequences. Specific strategies are warranted, focusing on mitigating statins’ underutilization and low potency. Additionally, consideration of adjunct therapies such as ezetimibe and bempedoic acid is crucial. Exploring novel agents like iPCSK9 or inclisiran emerges as promising avenues for safer and more effective alternatives in this population at high cardiovascular risk.

In Silico Assisted Identification, Synthesis, and In Vitro Pharmacological Characterization of Potent and Selective Blockers of the Epilepsy-Associated KCNT1 Channel.

Gain-of-function (GoF) variants in KCNT1 channels cause severe, drug-resistant forms of epilepsy. Quinidine is a known KCNT1 blocker, but its clinical use is limited due to severe drawbacks. To identify novel KCNT1 blockers, a homology model of human KCNT1 was built and used to screen an in-house library of compounds. Among the 20 molecules selected, five (CPK4, 13, 16, 18, and 20) showed strong KCNT1-blocking ability in an in vitro fluorescence-based assay. Patch-clamp experiments confirmed a higher KCNT1-blocking potency of these compounds when compared to quinidine, and their selectivity for KCNT1 over hERG and Kv7.2 channels. Among identified molecules, CPK20 displayed the highest metabolic stability; this compound also blocked KCNT2 currents, although with a lower potency, and counteracted GoF effects prompted by 2 recurrent epilepsy-causing KCNT1 variants (G288S and A934T). The present results provide solid rational basis for future design of novel compounds to counteract KCNT1-related neurological disorders.

The process of changing the potency of chromosomes, disease nosodes and organ preparations. How does this change the nature of the course of diseases and the physiological state of the subjects?

The work identified “active” and “inactive” chromosomes and how “inactive” chromosomes become “active”. For this purpose, a device for resonance diagnostics and therapy was used, connected to a computer, and the resonance of creation was used to transform inactive chromosomes into active ones. We added the sum of potencies to the already existing chromosome values. So, for example, at the beginning of the study, the patient was tested with a low potency of chromosomes equal to cassette No. 3 and ended with testing with cassette No. 10. Next, we “added”, connected the potency found in cassette No. 32 with high potencies to cassette No. 10. As a result, testing now began with cassette No. 1, and ended with cassette No. 12, i.e. testing increased to 4 cassettes. In a 50-year-old man, in the initial state, testing of chromosome potency began with cassette No. 18, and ended with cassette No. 31, i.e. 13 cassettes were tested. After connecting a cassette with a sufficiently high potency (cassette No. 47) to cassette No. 31, the start of testing changed and took place on cassette No. 2, and ended on cassette No. 49. Cassette testing increased to 49 cassettes, i.e. more than 3 times. Is it possible to potentiate not only chromosomes, but also other structures of the body? Potentiation is possible not only with chromosomes, but also with disease nosodes and organ preparations. An 84-year-old patient had untreated calculous cholecystitis from which he suffered. However, after transforming the nosode, after potentiating the nosode by connecting large cassettes to the nosode “calculous cholecystitis,” the patient’s calculous cholecystitis was extremely effectively cured. Treatment of an 82-year-old patient with cribriform prostate cancer. 10 years ago, the patient underwent surgery to remove cribriform prostate cancer - radical prostatectomy. After 10 years, a relapse occurred, which was treated with radiotherapy. The patient underwent 36 procedures, 5 procedures per week. At the end of the course of radiotherapy treatment, testing of cribriform prostate cancer showed that the cancer was still being tested, i.e. treatment has not been completed. We treated this patient using resonance therapy with changing, increasing potency of the nosode “caribous prostate cancer.” We increased the potency of the nosode in the patient to the point where the cancer could no longer be tested, i.e. We have completed treatment for cribriform prostate cancer. Potentiation is possible not only of chromosomes, nosodes, but also of organ preparations. Potentiation of the organopreparation “brain” was carried out in a 72-year-old subject who complained of general weakness, uncertainty of movements, memory loss, and poor sleep. After potentiation of the human brain organopreparation, the patient’s condition improved - he stopped complaining about changes in his physiological state. Thus, as a result of transformations of chromosomes, nosodes and organ preparations, as a result of their potentiation (increase in potency) when connecting a cassette of large potency, patients develop a new higher quality of chromosomes, nosodes and organ preparations. The effectiveness of the treatment of various diseases was achieved by us by increasing the potency of the nosode of the disease from which the patient suffered and using this potency for treatment. Potentiation of the human brain organopreparation significantly improved the physiological state of the subjects.