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Cardiac remodeling involves myocardial hypertrophy and fibrosis which impairs cardiac function and, ultimately, contributes to heart failure (HF) and mortality. Fibrosis largely develops due to excessive matrix deposition and lysyl oxidase(s)-dependent collagen cross-linking. In particular, lysyl oxidase-like 2 (LOXL2) has a critical role in disease progression, representing a promising therapeutic target and rationale for the development of novel, efficacious LOXL2 inhibitor(s). Herein, we describe the pre-clinical validation of a potent small molecule LOXL2 inhibitor as an anti-fibrotic agent, along with its clinical suitability, as high levels of target engagement were sustained in Phase 1 clinical trials while also being well tolerated. We show that LOXL2 concentration is increased in the plasma of patients with HF due to existing hypertension or aortic stenosis. Plasma LOXL2 concentration were correlated with the left ventricular mass index. A novel LOXL2 inhibitor, SNT-5382, was characterised, including in vitro and in vivo assessment of potency and mode of action, which showed beneficial drug-like properties. Preclinically, SNT-5382 reduced fibrosis and improved cardiac function in a myocardial infarction (MI) mouse model. Phase 1 clinical studies demonstrated a good safety and a PK profile capable of eliciting high and prolonged LOXL2 inhibition following repeated once daily oral dosing. Our findings underscore the pivotal role of LOXL2 in the development of HF. SNT-5382 exhibited potent anti-fibrotic efficacy in a MI model and sustained clinical target engagement. Trial registration : Australian New Zealand Clinical Trials Registry identifier: ACTRN12617001564347. Registered 21 November 2017- registered, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12617001564347

Dear Editor, In this study, measurement of target inhibition of lysyl oxidase-like 2 (LOXL2) in a high throughput manner from tissue lysates and blood was achieved by the tailored design of an activity-based probe (ABP), PXS-5878. Lysyl oxidases are a family of five enzymes critically responsible for the formation of cross-linked collagen and elastin, the hallmarks of fibrosis and stroma.1,2 One member in particular, LOXL2, has excellent pre-clinical target validation, is upregulated in various fibrotic diseases and cancer,3 and acts as a biomarker for disease severity and progression in humans.4,5 Despite overwhelming target rationale, the failure of the LOXL2 antibody simtuzumab to achieve positive clinical endpoints6,7 has undoubtedly hampered progress in the field and cast doubt over the validity of LOXL2 inhibition as a viable therapeutic approach. SEE PDF] In clinical studies with simtuzumab, the humanised version of AB0023, target engagement in human blood was not measured, triggering uncertainty about the lack of efficacy in humans.

Insulin-Regulated Aminopeptidase (IRAP) is an enzyme with important biological functions and the target of several drug-discovery efforts although no clinically useful inhibitors have been reported yet. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide and is active in blocking IRAP-dependent cross-presentation of an 8mer epitope. To better understand the mechanism of action and the basis for the unusual substrate selectivity of this inhibitor, we solved the crystal structure of the compound in complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the closed conformation previously observed with other low molecular weight inhibitors and hypothesized to be important for catalysis. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.

BackgroundImmune checkpoint inhibitors have transformed melanoma therapy but frequently cause immune-related adverse events (irAEs), including colitis, that limit treatment. Reliable biomarkers predicting toxicity remain lacking.MethodsIn this retrospective, multicenter study, we analyzed pretreatment serum samples from 331 patients with metastatic melanoma treated with anti-CTLA-4 (ipilimumab), anti-PD-1 (pembrolizumab or nivolumab), or combination ipilimumab/nivolumab. IgG autoantibody reactivity against 832 human protein antigens, including autoimmune targets, cytokines, tumor-associated antigens, and cancer pathway proteins, was profiled using multiplex bead-based arrays. Statistical analysis (Significance Analysis of Microarrays and Cox regression) identified autoantibody signatures associated with subsequent irAEs and immune-related colitis (ir-colitis).ResultsWe detected 47 autoantibodies predictive of irAEs, with KRT7, RPLP2, UBE2Z, and GPHN emerging as the strongest markers. Anti-KRT7 and anti-GPHN were specifically predictive in patients receiving PD-1 monotherapy, whereas anti-RPLP2 was associated with irAEs in ipilimumab/nivolumab combination therapy. For ir-colitis, 38 autoantibodies were identified, with five (PIAS3, RPLP0, UBE2Z, KRT7, and SDCBP) showing consistent predictive value across treatment groups. Anti-PIAS3 and anti-RPLP0 increased ir-colitis risk, while anti-SDCBP conferred protection. Notably, predictive profiles differed between PD-1-based and CTLA-4-based regimens, underscoring divergent mechanisms of toxicity. Several autoantibodies predictive of irAEs or ir-colitis also correlated with clinical outcome. ATG4D, MAGEB4, and IL4R were associated with prolonged progression-free and overall survival, whereas FGFR1 predicted both reduced irAE risk and inferior survival, consistent with the link between heightened immune activation, toxicity, and therapeutic benefit.ConclusionsThis study, to our knowledge, is the largest pretreatment autoantibody screen in melanoma immunotherapy, demonstrates that serum autoantibody profiles can stratify patients at risk for irAEs and ir-colitis. The identified signatures connect tumor-related and immunity-related antigens, stress-response pathways, and autoimmune mechanisms. Pretreatment autoantibody profiling offers a promising biomarker-driven approach for individualizing risk assessment, improving patient selection, and guiding early intervention strategies to enhance the safety of immune checkpoint blockade in melanoma. Beyond toxicity prediction, our findings also suggest that specific autoantibodies may reflect underlying immune activation states linked to therapeutic response.

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the 13 C-labelled metabolites that are generated by APC during catabolism of [U- 13 C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U- 13 C]2-oxoisovalerate, [U- 13 C]3-hydroxyisobutyrate, [U- 13 C]2-methylmalonate, [U- 13 C]isobutyrate, and [U- 13 C]propionate as well as several TCA cycle-dependent metabolites including lactate.

Isoleucine, together with leucine and valine, constitutes the group of branched-chain amino acids (BCAAs). BCAAs are transported from the blood into the brain parenchyma, where they can serve several distinct functions. Since brain tissue is known to oxidatively metabolize BCAAs to CO 2 , they are considered as fuel material in brain energy metabolism. Also, in the case of leucine, cultured astrocytes have been reported to be able to completely oxidize BCAA. While the metabolism of leucine by astroglia-rich primary culture (APC) has already been studied in detail, the metabolic fates of isoleucine and valine in these cells remained to be identified. Therefore, in the present study an NMR analysis was performed of 13 C-labelled metabolites generated in the catabolism of [U- 13 C]Ile by astrocytes and released by them into the incubation medium. APC potently removed isoleucine from the medium and metabolized it. The major isoleucine metabolites released from APC are 2-oxo-3-methylvalerate, 2-methylbutyrate, 3-hydroxy-2-methylbutyrate and propionate. To a lesser extent, APC generate and release also [2,3- 13 C]glutamine, [4,5- 13 C]glutamine and 13 C-labelled isotopomers of lactate and citrate. These results show that APC can release into the extracellular milieu catabolites and several TCA cycle dependent metabolites resulting from the degradation of isoleucine.

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a 13 C-nuclear magnetic resonance analysis was performed of [U- 13 C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media 13 C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a (13)C-nuclear magnetic resonance analysis was performed of [U-(13)C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media (13)C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a (13)C-nuclear magnetic resonance analysis was performed of [U-(13)C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media (13)C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.

Leucine is rapidly metabolized in astroglial primary cultures. Therefore, it is considered as valuable fuel in brain energy metabolism. Only few of the leucine metabolites generated and released by astroglial cells have been identified. Therefore, a more detailed study was performed analyzing by NMR techniques the 13C-labeled metabolites, which were released by astroglial primary cultures during the degradation of [U-(13)C]leucine. Confirming a former radioactive study this analysis revealed 13C-labeled 2-oxoisocaproate and ketone bodies. Additionally, 13C-labeled alanine, citrate, glutamine, lactate and succinate were identified. Their detailed isotopomer analysis proves that 13C-labeled acetyl-CoA enters the tricarboxylic acid cycle, that intermediates with a characteristic 13C-labeling pattern can be withdrawn at several positions of the cycle, and that in the case of lactate and alanine there appears to be a participation of an active phosphoenolpyruvate carboxykinase and/or malic enzyme pathway. Thus, astroglial cells generate and release into the extracellular fluid not only the leucine catabolites 2-oxoisocaproate and ketone bodies, but also several tricarboxylic acid cycle dependent metabolites.