Regeneration of the alveolar epithelium is necessary to restore tissue architecture and gas exchange capabilities in chronic pulmonary diseases such as fibrosing interstitial lung disease. While it is known alveolar type 2 (AT2) cells give rise to alveolar type 1 (AT1) cells to repair the alveolar epithelium after injury, methods to promote this process under pathological settings are poorly understood. Here, using a complex 3D organoid culture with TGF-β1 dependent impaired AT1 spheroid formation, we performed a high-throughput screen (HTS) with ∼16,800 compounds to identify small molecules that increase number of AT1 spheroids. Longitudinal single cell RNA sequencing (scRNA-seq) revealed that DB-11-BE87 increased AT1 regeneration by reducing TGF-β1 induced fibroblast activation, concurrently with AHR activation in those cells. These studies highlight a novel HTS system to identify factors that can promote AT1 differentiation and suggest AHR activation as a method to counteract pathological TGF-β1 signaling in pulmonary disease. ### Competing Interest Statement All authors are current or former employees and shareholders of Novartis. This study was funded by Novartis Biomedical Research.

The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.

SPPL2a (Signal Peptide Peptidase Like 2a) is an intramembrane aspartyl protease engaged in the function of B-cells and dendritic cells. Despite being an attractive target for modulation of the immune system, selective SPPL2a inhibitors are barely described in the literature. Recently, we have disclosed a selective, small molecular weight agent SPL-707 which confirmed that pharmacological inhibition of SPPL2a leads to the accumulation of its substrate CD74/p8 and as a consequence to a reduction in the number of B-cells as well as myeloid dendritic cells in mice. In this paper we describe the discovery of novel hydroxyethylamine based SPPL2a inhibitors. Starting from a rather lipophilic screening hit, several iterative optimization cycles allowed for its transformation into a highly potent and selective compound 15 (SPL-410) which inhibited in vivo CD74/p8 fragment processing in mice at 10 mg/kg oral dose.

A selective inhibitor of the kinetoplastid proteasome (GNF6702) is identified that is highly efficacious in vivo, clearing the parasites that cause leishmaniasis, Chagas disease and sleeping sickness from mice, highlighting the possibility of developing a single class of drugs for these neglected diseases. Chagas disease, leishmaniasis, and sleeping sickness are caused by the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively, and affect 20 million people worldwide. This study reports the results of a screen to find new conserved molecular targets and broad spectrum drugs that could be used to treat all three diseases. A selective inhibitor of the kinetoplastid proteasome (GNF6702) was identified as the most effective. It is highly efficacious in vivo, clearing parasites from mice in all three models of infection. GNF6702 is a non-competitive inhibitor, specific for kinetoplastid proteasome, and is well-tolerated in mice. These results highlight the possibility of developing a single class of drugs for these neglected diseases. Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually1. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target2. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Leishmaniasis is a parasitic disease that presents four main clinical syndromes: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), visceral leishmaniasis/kala azar (VL), and post kala azar dermal leishmaniasis (PKDL). Causative Leishmania are protozoan parasites that are transmitted among mammalian hosts by phlebotomine sandflies. In mammalian hosts, parasite cells proliferate inside the host phagocytic cells as round amastigotes. Infection of sandflies with Leishmania occurs during insect feeding on infected mammalian hosts. After introduction into the insect gut together with the blood meal, Leishmania amastigotes transform into elongated flagellated promastigotes that propagate in the insect gut. A new round of infection is initiated after the infected sandfly takes a blood meal from a naive mammalian host and introduces Leishmania parasites into the bite wound in the host dermis (Scheme 1). More than 20 different Leishmania species have been found to cause human leishmaniasis (Table 1). Leishmaniasis is endemic in 98 countries and is closely associated with poverty. More than a million new cases are reported per year and 350 million people are at risk of contracting the infection. For the most severe form of leishmaniasis, VL, ∼300 000 new cases are estimated to occur annually resulting in ∼40 000 deaths. Approximately 90% of all VL cases occur in 3 endemic foci: 1. India, Bangladesh, and Nepal; 2. East Africa; and 3. Brazil. In spite of the high prevalence, currently available treatments for leishmaniasis are inadequate. Pentavalent antimonials, the standard treatment for leishmaniasis for many decades, are not efficacious in Bihar (∼60% of VL cases worldwide) any longer due to widespread resistance to the drug in this region. Several new VL treatments have emerged during the past 10–15 years, but each has serious shortcomings (summarized in Table 2). These include paromomycin (injectable, long treatment, region-dependent efficacy), miltefosine (cost, teratogenicity, long treatment), and liposomal amphotericin B (cost, hospitalization, region-dependent efficacy). An additional challenge is represented by patients with HIV/VL coinfections who are more difficult to cure (lower initial and final cure rates), have greater susceptibility to drug toxicity, and have higher rates of death and relapse. Due to the limitations of the existing treatments, better drugs are urgently needed. Ideally, new VL drugs would be efficacious across all endemic regions, would affect cure in ≤10 days, and would cost <$10 per course (for a complete target product profile for new VL drugs, which was formulated by DNDi, see Table 4).1 Here we describe the disease history and parasite biology followed by a summary of the currently available treatments and, finally, review reports of novel small molecules with antileishmanial activity.

AbstractFor Abstract see ChemInform Abstract in Full Text.