Activin Receptor-like Kinase Research Articles

P0334 AGMB-129, an investigational ALK5 inhibitor for the treatment of Fibrostenosing Crohn’s Disease (FSCD), shows gastrointestinal (GI) restricted pharmacokinetics (PK) and a favorable safety profile in healthy subjects

Abstract Background AGMB-129 is an oral GI-restricted small molecule inhibitor of Activin Receptor-Like Kinase 5 (ALK5) intended for the treatment of Fibrostenosing Crohn’s Disease (FSCD). AGMB-129 potently blocks signaling of the key pro-fibrotic cytokine Transforming Growth Factor-b (TGFβ) and is designed to avoid clinically relevant systemic exposure via high first-pass metabolism to overcome the severe toxicities reported for prior systemic ALK5 inhibitors. This study evaluated the safety, systemic and ileal PK of single- and multiple-doses AGMB-129 in healthy subjects. Methods This was a randomized, double-blind, placebo-controlled, single and multiple ascending dose study in healthy subjects who received single doses (SD) up to 1200mg or multiple doses (MD) up to 400mg once-daily (QD) for 5 days or 200mg twice-daily (BID) or for 10 days, or placebo. The effect of food on PK of 400 mg SD was evaluated in a cross-over fashion. Results 82 participants (60 male/22 female) completed the study except for 2 subjects on placebo (both due to viral infection). After oral dosing AGMB-129 was rapidly absorbed with a median time to maximum plasma concentration (Tmax) of 0.7-1.5h, followed by a rapid decline in plasma concentration with geometric mean (gMean) concentration dropping <1 ng/mL within 4 to 8h post-dose resulting in low systemic exposure at all doses. The gMean maximum AGMB-129 plasma concentration (Cmax) after 1200mg SD was 63.3 ng/mL. After MD QD dosing, AGMB-129 accumulation based on the area-under-the-curve (AUC) was limited (accumulation ratio <2). After AGMB-129 200 mg BID, the gMean Cmax on Day 7 was 46.4ng/mL, and the gMean AGMB-129 concentration in ileal biopsies at 2h post-dose on Day 10 was 4244 ng/mL. MET-158 (inactive against ALK5) was the main circulating metabolite (gMean AUCMET-158/AUCAGMB-129 > 100). After intake with food, AGMB-129 Tmax was delayed by about 2h, Cmax and AUC increased by 1.3- and 2-fold, respectively, and intersubject variability was reduced compared to fasted intake. AGMB-129 was safe and well tolerated at all dosages. There were no dose-limiting toxicities, deaths, serious adverse events, or clinically-relevant abnormalities in laboratory results, vital signs or ECG parameters. Conclusion After oral dosing, low systemic and high ileal exposure to AGMB-129 was observed. This confirms the GI-restricted profile of AGMB-129 with systemic exposure more than 100-fold lower than previously observed for systemic ALK5 inhibitors [Rodón 2015, Jung 2020]. AGMB-129 was safe and well-tolerated at all dosages and is the first ALK5 inhibitor suitable for continuous daily dosing in humans. The STENOVA Ph2a study of AGMB-129 up to 200 mg BID in symptomatic FSCD is ongoing (NCT05843578). References Rodon J, Carducci M, Sepulveda-Sanchez JM, et al. Pharmacokinetic, pharmacodynamic and biomarker evaluation of transforming growth factor-β receptor I kinase inhibitor, galunisertib, in phase 1 study in patients with advanced cancer. Invest New Drugs (2015) 33:357-370. Jung SY, Hwang S, Clarke JM, et al. Pharmacokinetic characteristics of vactosertib, a new activin receptor-like kinase 5 inhibitor, in patients with advanced solid tumors in a first-in-human phase 1 study. Invest New Drugs (2020) 38:812-820.

An Activin Receptor-Like Kinase 1-governed monocytic lineage shapes an immunosuppressive landscape in breast cancer metastases.

The biology centered around the TGF-beta type I receptor Activin Receptor-Like Kinase (ALK)1 (encoded by ACVRL1) has been almost exclusively based on its reported endothelial expression pattern since its first functional characterization more than two decades ago. Here, in efforts to better define the therapeutic context in which to use ALK1 inhibitors, we uncover a population of tumor-associated macrophages (TAMs) that, by virtue of their unanticipated Acvrl1 expression, are effector targets for adjuvant anti-angiogenic immunotherapy in mouse models of metastatic breast cancer. The combinatorial benefit depended on ALK1-mediated modulation of the differentiation potential of bone marrow-derived granulocyte-macrophage progenitors, the release of CD14+ monocytes into circulation, and their eventual extravasation. Notably, ACVRL1+ TAMs coincided with an immunosuppressive phenotype, and were over-represented in human cancers progressing on therapy. Accordingly, breast cancer patients with a prominent ACVRL1hi TAM signature exhibited a significantly shorter survival. In conclusion, we shed light on an unexpected multimodal regulation of tumorigenic phenotypes by ALK1 and demonstrate its utility as a target for anti-angiogenic immunotherapy.

Transforming growth factor beta receptor type I (TGF-βRI) kinase inhibitors IOA-359 and IOA-360 stimulate erythropoiesis in MDS

Overactivation of the Transforming Growth Factor Beta (TGF-β) pathway is implicated in the pathogenesis of cytopenias in Myelodysplastic syndromes (MDS) and Acute Myeloid Leukemia (AML). IOA-359 and IOA-360 are potent small molecule inhibitors of the TGF-beta Receptor type I kinase (TGF-βRI, also referred to as ALK5, activin receptor-like kinase 5) that abrogate SMAD phosphorylation in hematopoietic cell lines. Both inhibitors were able to inhibit TGF-β mediated gene transcription at specific doses. ALK5 inhibitors abrogated the growth inhibitory effects of TGF-β on healthy hematopoietic stem cells and stimulated hematopoietic differentiation in cell lines and MDS/AML specimens. These data demonstrate preclinical efficacy of two novel ALK5 inhibitors, IOA-359 and IOA-360, in stimulating erythroid differentiation in MDS and AML.

Abstract A022: Discovery of conformationally constrained ALK2 inhibitors for the treatment of Diffuse Intrinsic Pontine Glioma (DIPG)

Abstract Despite considerable research on new therapeutics to treat DIPG, a universally fatal grade IV pediatric CNS tumor located in the pons region of the brainstem, there has been minimal progress. Somatic missense mutations in the bone morphogenetic protein (BMP) type I receptor ACVR1 gene encoding activin receptor-like kinase-2 (ALK2) are present in approximately 33% of children with DIPG, prompting our initial efforts towards the development of M4K2009, a 3,5-diphenylpyridine ALK2 inhibitor. Looking to improve potency, selectivity, and brain penetration, we subsequently designed, synthesized, and evaluated a first-in-class set of 5- to 7-member ether-linked and 7-member amine-linked conformationally constrained ALK2 inhibitors including M4K2308, M4K2281, M4K2304 and M4K2306. Overall, these new compounds are highly potent (ALK2 IC50 < 10 nM, nanoBRET ALK2 IC50 < 20 nM), selective (>100-fold over ALK5), brain-penetrant (Cbrain,4h/Cplasma,4h > 3, 10 mg/kg, NOD-SCID male mice) and show in vivo exposure (Cplasma,6h = 1−2 µM, 25 mg/kg, NOD-SCID male mice). In particular, M4K2308 displayed a good balance of potency and DMPK properties and is currently undergoing preclinical evaluation by M4K Pharma. Citation Format: David Smil, Héctor González-Álvarez, Deeba Ensan, Jong Fu Wong, Julien Cros, Laurent Hoffer, Taira Kiyota, Brian J. Wilson, Carlos A. Zepeda-Velázquez, Ahmed Aman, Methvin B. Isaac, Tao Xin, Alex N. Bullock, Peter B. Sampson, Rima Al-awar. Discovery of conformationally constrained ALK2 inhibitors for the treatment of Diffuse Intrinsic Pontine Glioma (DIPG) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A022.

Pharmacokinetics of Zilurgisertib With and Without Food from Single and Multiple Ascending Dose Phase 1 Studies in Healthy Adults.

The oral, potent, and highly selective activin receptor-like kinase 2 (ALK2) inhibitor zilurgisertib (INCB000928) is in development as a treatment for fibrodysplasia ossificans progressiva (FOP), and for anemia due to myelofibrosis, myelodysplastic syndromes, and multiple myeloma. Saliva is an attractive alternative to blood for drug monitoring and pharmacokinetic analysis, as it is non-invasive to retrieve. This is beneficial for patients, such as those with FOP, for whom blood draws can be challenging due to soft tissue damage susceptibility that can cause progressive heterotopic ossification, and for whom tourniquet time and blood draws must be minimized. The objectives of these studies were to evaluate zilurgisertib pharmacokinetics, safety, tolerability, and the effect of food in healthy participants from phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) studies. Both the SAD and MAD studies were double-blind, randomized, placebo-controlled dose escalation studies. In the SAD study, healthy participants received a single oral dose of zilurgisertib (10, 25, 50, 100, 175, 250, or 500 mg) or placebo in the fasted state. A further group of healthy participants were enrolled into an additional "food effect" cohort and randomized to receive a single oral dose of zilurgisertib (100 mg) after either an overnight fast or a high-fat meal in a 2-way crossover manner. In the MAD study, healthy participants received oral zilurgisertib at 50, 100, 150, 200, or 400 mg once daily or 300 mg twice daily in the fasted state. Blood, saliva, and urine samples were collected for zilurgisertib pharmacokinetic analysis. Safety was assessed throughout both studies. Overall, 91 participants (70 active, 21 placebo) were enrolled and randomized to the SAD study and 79 participants (59 active, 20 placebo) were enrolled and randomized to the MAD study. Zilurgisertib was generally well tolerated, and adverse events were generally of mild-to-moderate severity. Zilurgisertib was rapidly absorbed, with median time to maximum plasma drug concentration (Cmax) of 2.0-4.1 h post-dose. Zilurgisertib exposure was more than dose proportional after single and multiple doses over the dose range tested, suggesting non-linear pharmacokinetics. Plasma half-life values ranged from 22.8 to 31.4 h, supporting once-daily dosing. There was a strong correlation between zilurgisertib concentrations in saliva and plasma. No food effect was observed on zilurgisertib pharmacokinetics, with geometric mean ratio (90% confidence interval) Cmax and area under the plasma concentration-time curve values of 0.98 (0.91to1.06) and 1.03 (0.97to1.10). Renal excretion under fasted conditions was 16% and 27% of total drug clearance with single and multiple doses, respectively; therefore, it was not the predominant pathway for zilurgisertib elimination. Zilurgisertib exhibited a favorable pharmacokinetic profile amenable to once-daily dosing that can be administered without regard to food. Study results support further clinical development of zilurgisertib in patients.

Arterial endothelial deletion of hereditary hemorrhagic telangiectasia 2/ Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder with manifestations including severe nose bleeding and microhemorrhage in brains. Despite being the second most common inherited bleeding disorder, the pathophysiological mechanism underlying HHT-associated hemorrhage is poorly understood. HHT pathogenesis is thought to follow a Knudsonian two-hit model, requiring a second somatic mutation for lesion formation. Mutations in activin receptor-like kinase 1 ( ALK1 ) gene cause HHT type 2. We hypothesize that somatic mutation of Alk1 in arterial endothelial cells (AECs) leads to arterial defects and hemorrhage. Here, we mutated Alk1 in AECs in postnatal mice using Bmx(PAC)-Cre ERT2 and found that somatic arterial endothelial mutation of Alk1 was sufficient to induce spontaneous epistaxis and multifocal cerebral microhemorrhage. This bleeding occurred in the presence of tortuous and enlarged blood vessels, loss of arterial molecular marker Efnb2 , disorganization of vascular smooth muscle, and impaired vasoregulation. Our data suggest that arterial endothelial deletion of Alk1 leading to reduced arterial identity and disrupted vascular smooth muscle cell coverage is a plausible molecular mechanism for HHT-associated severe epistaxis. This work provides the first evidence that somatic Alk1 mutation in AECs can cause hemorrhagic vascular lesions, offering a novel preclinical model critically needed for studying HHT-associated epistaxis, and delineating an arterial mechanism to HHT pathophysiology.

PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder characterized by arteriovenous malformations (AVMs). Loss-of-function mutations in Activin receptor-like kinase 1 (ALK1) cause type 2 HHT and Alk1 knockout (KO) mice develop AVMs due to overactivation of VEGFR2/PI3K/AKT signaling pathways. However, the full spectrum of signaling alterations in Alk1 mutants remains unknown and means to combat AVM formation in patients are yet to be developed. Single-cell RNA sequencing of endothelial-specific Alk1 KO mouse retinas and controls identified a cluster of endothelial cells (ECs) that was unique to Alk1 mutants and that overexpressed fluid shear stress (FSS) signaling signatures including upregulation of the mechanosensitive ion channel PIEZO1. PIEZO1 overexpression was confirmed in human HHT lesions, and genetic and pharmacological PIEZO1 inhibition was tested in Alk1 KO mice, as well as downstream PIEZO1 signaling. Pharmacological PIEZO1 inhibition, and genetic Piezo1 deletion in Alk1 -deficient mice effectively mitigated AVM formation. Furthermore, we identified that elevated VEGFR2/AKT, ERK5-p62-KLF4, hypoxia and proliferation signaling were significantly reduced in Alk1 - Piezo1 double ECKO mice. PIEZO1 overexpression and signaling is integral to HHT2, and PIEZO1 blockade reduces AVM formation and alleviates cellular and molecular hallmarks of ALK1-deficient cells. This finding provides new insights into the mechanistic underpinnings of ALK1-related vascular diseases and identifies potential therapeutic targets to prevent AVMs.

CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia.

Increased endothelial cell proliferation is a hallmark of arteriovenous malformations (AVMs) in hereditary hemorrhagic telangiectasia (HHT). Here, we report a cyclin-dependent kinase 6 (CDK6)-driven mechanism of cell cycle deregulation involved in endothelial cell proliferation and HHT pathology. Specifically, endothelial cells from the livers of HHT mice bypassed the G1/S checkpoint and progressed through the cell cycle at an accelerated pace. Phosphorylated retinoblastoma (pRB1)-a marker of G1/S transition through the restriction point-accumulated in endothelial cells from retinal AVMs of HHT mice and endothelial cells from skin telangiectasia samples from HHT patients. Mechanistically, inhibition of activin receptor-like kinase 1 signaling increased key restriction point mediators, and treatment with the CDK4/6 inhibitors palbociclib or ribociclib blocked increases in pRB1 and retinal AVMs in HHT mice. Palbociclib also improved vascular pathology in the brain and liver, and slowed cell cycle progression in endothelial cells and endothelial cell proliferation. Endothelial cell-specific deletion of CDK6 was sufficient to protect HHT mice from AVM pathology. Thus, clinically approved CDK4/6 inhibitors might have the potential to be repurposed for HHT.

Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions.

Background/Objectives: Cripto-1 (CR1) is a plurifunctional embryonic protein required for implantation and re-expressed in the adult during wound repair, inflammation, and tumorigenesis. CR1 and its predicted CR1 pseudogene product Cripto-3/CR3 are highly homologous proteins, and given this physical attribute, commercially available antibodies cannot discriminate between CR1 and CR3. Methods: A series of mouse monoclonal antibodies [MoAbs] were developed with a high-affinity binding that can differentiate human CR1/CR3 proteins and showed no measurable cross-reactivity. Results: Using these reagents, we confirm that CR3 is a bona fide translated protein found in human tumor tissue, cancer cell lysates, and in normal/cancer patient donor sera. We also reveal that CR1 and CR3 compete for binding to signal transduction protein Nodal, glucose-regulated protein 78Da (GRP78), and activin receptor-like kinase 4 (Alk4). Our discriminatory MoAbs provide new reagents to help clarify current CR1/CR3 protein expression vagaries in the Cripto field of study, challenging established CR1 conventions. In addition, our data validate CR3 involvement in human carcinogenesis and cell signaling pathways, with potential clinical relevance in determining cancer patient prognosis and disease severity.

Arterial-Lymphatic-Like Endothelial Cells Appear in Hereditary Hemorrhagic Telangiectasia 2 and Contribute to Vascular Leakage and Arteriovenous Malformations.

Arteriovenous malformations (AVMs) are characteristic of hereditary hemorrhagic telangiectasia. Loss-of-function mutations in the activin receptor-like kinase 1 (Alk1) are linked to hemorrhagic telangiectasia type 2. Endothelial-specific deletion of Alk1, endothelial lineage tracing, transcriptomics of single-cell analysis, and electron microscopy were performed to examine the vascular phenotype and characteristics of ALK1-deficient endothelial cells (ECs) after EC-specific Alk1 deletion. Ischemia assays were used to examine the cell capacity for vascular malformation. Connectivity Map with transcriptomic analysis was applied to identify chemical compounds. Specific methods for arteriovenous malformations, such as micro-computed tomography, with other molecular and cell biological tools were also performed. We performed endothelial-specific deletion of Alk1 in mice and found severe arteriovenous malformations and vascular leakage. The transcriptomics of single-cell analysis revealed a new distinctive cell cluster formed after Alk1 deletion where the cells coexpressed arterial and lymphatic endothelial markers. The analysis projected that these cells potentially originated from arterial ECs after Alk1 deletion. This new population was referred to as arterial-lymphatic-like ECs according to its cellular markers, and its appearance was validated in the pulmonary small arteries after Alk1 deletion. Transplantation of these cells caused vascular malformations. Endothelial lineage tracing confirmed that these new arterial-lymphatic-like ECs were derived from ALK1 depleted ECs, potentially arterial ECs. We discovered that SOX17 (SRY-box transcription factor 17) induction was responsible for the derivation of these arterial-lymphatic-like ECs. We showed that direct binding of MDM2 (mouse double minute 2) was required for Sox17 to execute this activity. Inhibition of MDM2 reduced the arteriovenous malformations in the mouse model. Together, our studies revealed the mechanistic underpinnings of ALK1 signaling in regulating the endothelial phenotype and provided possibilities for new therapeutic strategies in hemorrhagic telangiectasia type 2.

IL-1β Induces LDL Transcytosis by a Novel Pathway Involving LDLR and Rab27a.

In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial (Canakinumab Antiinflammatory Thrombosis Outcome Study) treated advanced atherosclerosis using a blocking antibody for IL-1β (interleukin-1β); this significantly reduced cardiovascular events. However, whether IL-1β regulates early disease, particularly LDL transcytosis, remains unknown. We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1β. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1β and LDL to visualize acute LDL deposition in the aortic arch. Exposure to picomolar concentrations of IL-1β induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1β increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1β on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNA sequencing data to curate a list of Rab (Ras-associated binding) GTPases affected by IL-1β, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1β. This was phenocopied by depletion of the Rab27a effector JFC1 (synaptotagmin-like protein 1). In vivo, IL-1β increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1β-induced LDL accumulation in the aorta. IL-1β induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1β may contribute to the acceleration of early disease.

Synthesis and biological evaluation of sulfonamide derivatives containing imidazole moiety as ALK5 inhibitors.

Four series of sulfonamide derivatives (13a-b, 14a-d, 15a-b, and 16a-d) were synthesized and evaluated for their activin receptor-like kinase 5 (ALK5) inhibitory activities. Of these, compounds 13b (IC50 = 0.130μM) and 15a (IC50 = 0.130μM) showed the highest inhibitory activities against ALK5 kinase, with activities similar to the positive control LY-2157299. Notably, we discovered that introduction of sulfonamide group at the 2-position of the central imidazole ring significantly increased ALK5 inhibitory activity. Compounds 13b and 15a did not show toxicity in A549 cells up to the maximum concentration of 50μM, and effectively inhibited TGF-β1-induced Smad-signaling and cell motility in A549 cells. The results indicate that compounds 13b and 15a are worth of further development as anticancer agents.

Loss of TGFβ-Mediated Repression of Angiopoietin-2 in Pericytes Underlies Germinal Matrix Hemorrhage Pathogenesis.

TGF (transforming growth factor)-β pathway is central to blood-brain barrier development as it regulates cross talk between pericytes and endothelial cells. Murine embryos lacking TGFβ receptor Alk5 (activin receptor-like kinase 5) in brain pericytes (mutants) display endothelial cell hyperproliferation, abnormal vessel morphology, and gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH), leading to perinatal lethality. Mechanisms underlying how ALK5 signaling in pericytes noncell autonomously regulates endothelial cell behavior remain elusive. Transcriptomic analysis of human brain pericytes with ALK5 silencing identified differential gene expression. Brain vascular cells isolated from mutant embryonic mice with GMH-IVH and preterm human IVH brain samples were utilized for target validation. Finally, pharmacological and genetic inhibition was used to study the therapeutic effects on GMH-IVH pathology. Herein, we establish that the TGFβ/ALK5 pathway robustly represses ANGPT2 (angiopoietin-2) in pericytes via epigenetic remodeling. TGFβ-driven SMAD (suppressor of mothers against decapentaplegic) 3/4 associates with TGIF1 (TGFβ-induced factor homeobox 1) and HDAC (histone deacetylase) 5 to form a corepressor complex at the Angpt2 promoter, resulting in promoter deacetylation and gene repression. Moreover, murine and human germinal matrix vessels display increased ANGPT2 expression during GMH-IVH. Isolation of vascular cells from murine germinal matrix identifies pericytes as a cellular source of excessive ANGPT2. In addition, mutant endothelial cells exhibit higher phosphorylated TIE2 (tyrosine protein kinase receptor). Pharmacological or genetic inhibition of ANGPT2 in mutants improves germinal matrix vessel morphology and attenuates GMH pathogenesis. Importantly, genetic ablation of Angpt2 in mutant pericytes prevents perinatal lethality, prolonging survival. This study demonstrates that TGFβ-mediated ANGPT2 repression in pericytes is critical for maintaining blood-brain barrier integrity and identifies pericyte-derived ANGPT2 as an important pathological target for GMH-IVH.

Discovery of Two Highly Selective Structurally Orthogonal Chemical Probes for Activin Receptor-like Kinases 1 and 2.

Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.

TGFβ signaling pathway is altered by HLA-B27 expression, resulting in pathogenic consequences relevant for spondyloarthritis

BackgroundAssociation of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years, but still remains unexplained. We recently showed that HLA-B27 expressed in wing imaginal disc from HLA-B27/human-β2 microglobulin (hβ2m) transgenic Drosophila deregulated bone morphogenetic protein (BMP) pathway by interacting physically with type I BMP receptor (BMPR1) Saxophone (Sax), leading to crossveinless phenotype.MethodsGenetic interaction was studied between activin/transforming growth factor β (TGFβ) pathway and HLA-B27/hβ2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs. In mesenteric lymph node (mLN) T cells from HLA-B27/hβ2m rat (B27 rat), physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s, phosphorylation of small mothers against decapentaplegic (SMADs) and proteins of the non-canonical BMP/TGFβ pathways induced by its ligands, and the transcript level of target genes of the TGFβ pathway, were evaluated.ResultsIn HLA-B27/hβ2m transgenic Drosophila, inappropriate signalling through the activin/TGFβ pathway, involving Baboon (Babo), the type I activin/TGFβ receptor, contributed to the crossveinless phenotype, in addition to deregulated BMP pathway. We identified peptides bound to HLA-B27 with the canonical binding motif in HLA-B27/hβ2m transgenic Drosophila wing imaginal disc. We demonstrated specific physical interaction, between HLA-B27/hβ2m and mammalian orthologs of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGFβ receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFβ1 was increased in T cells from B27 rats, showing evidence for deregulated TGFβ pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFβ exposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly, Tgfb1 expression was reduced in naive T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern.ConclusionsThis study shows that HLA-B27 alters the TGFβ pathways in Drosophila and B27 rat. Given the importance of this pathway in CD4 + T cells differentiation and regulation, its disturbance could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and altered regulatory T cell phenotype observed in B27 rats.

Activin E upregulates uncoupling protein 1 and fibroblast growth factor 21 in brown adipocytes

Activin E activates brown and beige adipocytes and has been controversially implicated as a factor that induces obesity and fatty liver. Here, we sought to address this controversial issue by producing recombinant human activin E to evaluate its effects on HB2 brown adipocytes in vitro. Activin E increased uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) mRNA expression in the adipocytes. This upregulation was suppressed by SB431542, an inhibitor of activin receptor-like kinase (Alk) TGF-β type I receptors. SB431542 also inhibited the activin E-induced phosphorylation of Smad2/3. A promoter assay using a CAGA-Luc reporter and Alk expression vectors revealed that activin E activated the TGF-β/activin pathway via Alk7. The upregulation of Ucp1 and Fgf21 mRNA might be mediated through Alk7 and Smad2/3 phosphorylation. Activin E is a potential stimulator of energy expenditure by activating brown adipocytes and highlights its potential as a therapeutic target for treating obesity.

Fibrotic remodeling in joint diseases: induction and inhibition of fibrosis in fibroblast-like synoviocytes

BackgroundWe aimed to investigate the development of synovial fibrosis in vitro and how the fibrosis can be halted. Synovial fibrosis causes joint stiffness in arthritic diseases. The pathway of the fibrotic growth factor, transforming growth factor-beta (TGF-β), has been associated with joint pain in osteoarthritis (OA) and with the fibroid phenotype of rheumatoid arthritis (RA). This suggests that synovial fibrosis, thus accumulation of extracellular matrix (ECM) proteins, plays a role in the clinical manifestations of the diseases. Improving our understanding of fibrotic development may aid in selecting appropriate treatments and development of drugs that can target synovial fibrosis.MethodsWe isolated primary fibroblast-like synoviocytes (FLS) from the synovial membrane of patients undergoing total knee replacement surgery. To investigate the development of synovial fibrosis, the FLS were cultured in a crowded in vitro model mimicking the ECM. TGF-β1 was used as the fibrotic initiator, the activin receptor-like kinase 5 inhibitor (ALK5i), the anti-fibrotic drug nintedanib, and the anti-inflammatory drug tofacitinib were used as fibrotic inhibitors. The ECM protein formation was quantified in the conditioned media using specific biomarkers of type I, III, and VI collagen formation and fibronectin turnover.ResultsThe TGF-β stimulation inducted fibrogenesis by increasing the biomarkers of fibronectin turnover, type I, III, and VI collagen formation. ALK5i and nintedanib inhibited the TGF-β response across all biomarkers. Tofacitinib trended towards inhibiting TGF-β response with up to 78% inhibition. All the treatments preserved cell viability.ConclusionWe have established an in vitro model for assessing fibrogenesis in primary FLS, which can be used to assess the anti-fibrotic effect of multiple drug types. Our study implies that synovial fibrosis can be induced by TGF-β, which additionally can be halted by both direct and indirect inhibition with anti-fibrotic substances. The anti-inflammatory drug tofacitinib also halted the fibrogenesis to some extent; thus, it may exert an anti-fibrotic effect.

An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Abstract 115: Scavenger Receptor-BI Mediates Endothelial Uptake Of Lipoprotein(a)

Elevated levels of lipoprotein(a) [Lp(a)], an apoB100-containing lipoprotein, are an independent risk factor for atherosclerotic cardiovascular disease, even in the setting of effective reduction of plasma low-density lipoprotein (LDL) cholesterol. To date, the mechanisms whereby Lp(a) exerts its atherogenic effects remain poorly understood, and no treatments aimed at lowering Lp(a) have been approved. Two receptors - scavenger receptor-BI (SR-BI) and activin receptor-like kinase 1 (ALK1) - have been proposed to bind apoB100 and mediate the atherogenic uptake and transcytosis of LDL across the endothelial cell (EC) barrier. EC-specific SR-BI knockout reduces atherosclerosis in both apoE and LDLR knockout mice. We have shown that SR-BI also mediates EC uptake of nascent chylomicrons. Although binding of ALK1 to apoB is believed to occur through the N-terminus of apoB, the receptor is not involved in the uptake of chylomicrons. We assessed which if any of these receptors are involved in Lp(a) uptake by ECs. Despite the presence of apoB100, EC uptake of Lp(a), like apoB48 chylomicrons, was reduced by SR-BI, but not ALK1, knockdown. We then used a series of small N terminal apoB peptides (apoB3-apoB18, with sequences comprising the 3%, 8%, 12%, 15%, and 18% N terminus of apoB) to compete with Lp(a) uptake. Both chylomicron and Lp(a) uptake was inhibited by apoB15 and apoB18, whereas LDL uptake was additionally inhibited by apoB12. These observations suggest that a) the binding sequence to SR-BI is between residues 547-684 of apoB, and b) an additional sequence between residues 365-547 participates in apoB binding to ALK1. Our findings suggest that apo(a) alters the structure of apoB to make it a better ligand for SR-BI uptake by ECs. Because this uptake pathway leads to transcytosis, we hypothesize that the greater atherogenicity of Lp(a) is mediated by its increased binding to and uptake by ECs.

Synthesis of amide derivatives containing the imidazole moiety and evaluation of their anti-cardiac fibrosis activity.

Three series of N-{[4-([1,2,4]triazolo[1,5-α]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl]methyl}acetamides (14a-d, 15a-n, and 16a-f) were synthesized and evaluated for activin receptor-like kinase 5 (ALK5) inhibitory activities in an enzymatic assay. The target compounds showed high ALK5 inhibitory activity and selectivity. The half maximal inhibitory concentration (IC50) for phosphorylation of ALK5 of 16f (9.1 nM), the most potent compound, was 2.7 times that of the clinical candidate EW-7197 (vactosertib) and 14 times that of the clinical candidate LY-2157299. The selectivity index of 16f against p38α mitogen-activated protein kinase was >109, which was much higher than that of positive controls (EW-7197: >41, and LY-2157299: 4). Furthermore, a molecular docking study provided the interaction modes between the target compounds and ALK5. Compounds 14c, 14d, and 16f effectively inhibited the protein expression of α-smooth muscle actin (α-SMA), collagen I, and tissue inhibitor of metalloproteinase 1 (TIMP-1)/matrix metalloproteinase 13 (MMP-13) in transforming growth factor-β-induced human umbilical vein endothelial cells. Compounds 14c and 16f showed especially high activity at low concentrations, which suggests that these compounds could inhibit myocardial cell fibrosis. Compounds 14c, 14d, and 16f are potential preclinical candidates for the treatment of cardiac fibrosis.