Abstract Extracellular 2',3'-cyclic GMP-AMP (cGAMP) has a critical immune-transmitter role in the tumor microenvironment (TME). Tumor cells produce and secrete cGAMP, which primes immune cells for tumor rejection through STING (stimulator of interferon genes) signalling (Marcus et al., Immunity 2018; Carozza et al., Nature Cancer 2020). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) is the only enzyme known to hydrolyze extracellular cGAMP. In concert with the ectoATPase CD39 and the 5'-nucleotidase CD73, ENPP1 also contributes to the generation of an immunosuppressive TME by converting extracellular ATP into adenosine. ENPP1 is overexpressed in a number of tumor types, including breast cancer, liver cancer, thyroid cancer and sarcomas, and has been associated with poor outcome. Hence, inhibition of ENPP1 is an emerging strategy to augment anti-tumor immunity by stabilizing extracellular cGAMP and ATP, thereby turning cold tumors into immunologically hot tumors. Here, we report on the identification and characterization of novel chemical series of ENPP1 inhibitors. A drug-like small-molecule library (~160,000 compounds) was screened to identify inhibitors of ENPP1-mediated cGAMP hydrolysis. Hits were confirmed to also inhibit ATP hydrolysis by ENPP1, but not nucleotide hydrolysis mediated by the closely related ENPP2 (autotaxin) enzyme. A selection of hits, chemically distinct from previously reported ENPP1 inhibitors (such as those based on QS1; Carroza et al, Cell Chem Biol, 2020), were subjected to hit-to-lead optimization supported by structure-based guidance. This led to the identification of ENPP1 inhibitors with sub-nM potency on ENPP1 in biochemical assays, which maintained a >1000x selectivity window with respect to ENPP2 and to other phosphodiesterases. These compounds translated well to stabilization of nucleotides in the presence of ENPP1-overexpressing cancer cell lines, with IC50 values in the nM range. A selection of compounds was profiled for DMPK (drug metabolism and pharmacokinetic) parameters, and compounds with a suitable profile were prioritized for in vivo evaluation. Orally bio-available, metabolically stable compounds were assessed in mouse syngeneic tumor models, selected on the basis of high ENPP1 and high cGAS (cGAMP synthase) expression. Stabilization of cGAMP, activation of a STING-mediated cytokine response and immune-cell infiltration/activation in the TME were used as pharmacodynamic endpoints. An update on in vivo efficacy data in a range of ENPP1-positive tumor models will be provided during the presentation. These novel orally bioavailable ENPP1 inhibitors unleash local, TME-restricted, innate immune activation, and hold the promise to overcome the current limitations of direct STING agonists. Citation Format: Matthias Versele, Javier del Pino Garcia, Ilse Vandecaetsbeek, Karolien Castermans, Ranie Kellens, Wanda Haeck, Hugo Klaassen, Arnaud Bourin, Dries De Clercq, Sara Allasia, Sandro Boland, Arnaud Marchand, Patrick Chaltin, Mathieu Bollen. Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 53.

IBD patients have an increasing number of treatment options to deal with inflammation in the GI tract but these anti-inflammatory agents are not able to prevent deleterious tissue remodelling that results in scaring and stenosis formation. ROCK is a serine–threonine kinase that has been shown to be instrumental in fibrosis, facilitating signalling between several pro-fibrotic receptors and involved in myofibroblast differentiation. However, the development of therapeutics targeting ROCK has been limited as systemic exposure of ROCK inhibitors also results in vasodilation of venous smooth muscles and hypotension. Redx are developing GI targeted soft ROCK inhibitors that potently inhibit ROCK activity in the GI tract but are rapidly metabolised in plasma which limits systemic exposure and avoids the safety concerns of systemic inhibition of ROCK activity. To assess the potential of the soft ROCK1/2 inhibitor REDX08087 as an anti-fibrotic treatment the adoptive T-cell transfer was utilised. CD4+CD25-CD62L+ naïve T cells were injected i.p. in CB-17 SCID mice on day 0 (adoptive transfer, AT). Mice developed symptoms of colitis at week 2 and beyond, at which point therapy was initiated. Mice were treated with 25 mg/kg anti-TNF i.p. SID or with a combination of anti-TNF i.p. and 3 mg/kg REDX08087 via oral gavage QD (n = 10 in each group). Placebo-treated mice received 25 mg/kg/day IgG1 i.p. and vehicle p.o. To assess whether REDX08087 could reverse established fibrosis the mouse chronic DSS model was utilised. Intestinal fibrosis was induced by administering 2.5% DSS in the drinking water for 1 week, followed by 2 weeks of normal drinking water. This cycle was repeated three times, during which mice were treated with 3 mg/kg or 10 mg/kg of REDX08087 via oral gavage QD from week 7, groups of mice were sacrificed at 6, 9, and 12 weeks after their first exposure to DSS. The GI restricted soft ROCK1/2 inhibitor REDX08087 was able to significantly prevent the development of fibrosis in an adoptive T-cell transfer model of IBD when dosed at 3 mg/kg qd for 20 days. In addition, a trend for an increased anti-fibrotic effect was observed with concomitant treatment with an anti-TNFα. Furthermore, the soft ROCK1/2 inhibitor REDX08087 was able to reverse fibrosis in a 12-week chronic DSS model of IBD when dosed from week 6 to 12 at 10 mg/kg. These data suggest that GI restricted soft ROCK inhibitors will be able to deliver clinical efficacy and reduce stenosis formation in IBD patients. The effect of the compounds will be delivered in a targeted fashion in the GI tract and will avoid the CV toxicities observed in other ROCK inhibitors.

BACKGROUND: Intestinal fibrosis resulting in (sub) obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFb-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFb1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Inhibitors of LIM kinases are considered of interest for several indications, including elevated intraocular pressure (IOP), cancer, or infection by HIV-1. LX-7101 (Lexicon Pharmaceuticals) was advanced to Phase-I clinical trials as an IOP-lowering agent for treatment of glaucoma. We here discuss the design, synthesis and evaluation of LIMK inhibitors based on a pyrrolopyrimidine scaffold, which represent close analogs of LX-7101. Exploration of structure-activity relationships revealed that many of such compounds, including LX-7101, cause potent inhibition of LIMK1 and LIMK2, and also ROCK2 and PKA. Molecular variations around the various structural elements of LX-7101 were attempted. Substitution on position 6 of the pyrrolopyrimidine scaffold led to the identification of LX-7101 analogs displaying good selectivity versus ROCK, PKA and Akt.

Extensive structure–activity studies on three different modification sites resulted in a series of LIM kinase inhibitors, containing a novel tricyclic hinge-binding motif based on the pyrrolopyrimidine scaffold.

ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed.

PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-associated side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors containing a γ-lactone structure. Such molecules are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors were highly active on both PDE4B1 and PDE4D2 and underwent rapid degradation in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC50 = 2.5 nM) with rapid inactivation in plasma (t1/2 <5′). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.

The dominant strategy for discovery of new antimalarial drugs relies on cell-free assays on specific biochemical pathways of Plasmodium falciparum . However, it appears that screening directly on the parasite is a more rewarding approach. The "drug to genome to drug" approach consists of testing a small set of structural analogues of a drug acting on human proteins that have plasmodial orthologues. Both man and plasmodium possess cyclic nucleotide phosphodiesterases (PDEs) that are key players of cell homeostasis. We synthesized and tested 40 analogues of tadalafil, a human PDE5 inhibitor, on P. falciparum in culture and obtained potent inhibitors of parasite growth. We discuss the structure-activity relationships, which support the hypothesis that our compounds kill the parasite via inhibition of plasmodial PDE activity. We also prove that antiplasmodial derivatives inhibit the hydrolysis of cyclic nucleotides of the parasite, validating the cAMP/cGMP pathways as therapeutic targets against Plasmodium falciparum.