SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late-onset Alzheimer's disease.

INTRODUCTION: The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. METHODS: We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. RESULTS: SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. DISCUSSION: 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights: Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.

Identification of ß-Lactams Active against Mycobacterium tuberculosis by a Consortium of Pharmaceutical Companies and Academic Institutions.

Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of ß-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 ß-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of ß-lactams screened were active against Mtb, many without a ß-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against Mycobacterium tuberculosis.

The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis. Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC90 < 5 µM). We conducted a structure-activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC90) of 4.9 µM with no cytotoxicity (IC50 > 100 µM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features.

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

Identification of tyrosine kinase inhibitors that halt Plasmodium falciparum parasitemia.

Although current malaria therapies inhibit pathways encoded in the parasite's genome, we have looked for anti-malaria drugs that can target an erythrocyte component because development of drug resistance might be suppressed if the parasite cannot mutate the drug's target. In search for such erythrocyte targets, we noted that human erythrocytes express tyrosine kinases, whereas the Plasmodium falciparum genome encodes no obvious tyrosine kinases. We therefore screened a library of tyrosine kinase inhibitors from Eli Lilly and Co. in a search for inhibitors with possible antimalarial activity. We report that although most tyrosine kinase inhibitors exerted no effect on parasite survival, a subset of tyrosine kinase inhibitors displayed potent anti-malarial activity. Moreover, all inhibitors found to block tyrosine phosphorylation of band 3 specifically suppressed P. falciparum survival at the parasite egress stage of its intra-erythrocyte life cycle. Conversely, tyrosine kinase inhibitors that failed to block band 3 tyrosine phosphorylation but still terminated the parasitemia were observed to halt parasite proliferation at other stages of the parasite's life cycle. Taken together these results suggest that certain erythrocyte tyrosine kinases may be important to P. falciparum maturation and that inhibitors that block these kinases may contribute to novel therapies for P. falciparum malaria.

Inhibition of Sphingosine Phosphate Receptor 1 Signaling Enhances the Efficacy of VEGF Receptor Inhibition.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Identification of Compounds with pH-Dependent Bactericidal Activity against Mycobacterium tuberculosis.

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.

Imidazopyridine Compounds Inhibit Mycobacterial Growth by Depleting ATP Levels.

The imidazopyridines are a promising new class of antitubercular agents with potent activity in vitro and in vivo We isolated mutants of Mycobacterium tuberculosis resistant to a representative imidazopyridine; the mutants had large shifts (>20-fold) in MIC. Whole-genome sequencing revealed mutations in Rv1339, a hypothetical protein of unknown function. We isolated mutants resistant to three further compounds from the series; resistant mutants isolated from two of the compounds had single nucleotide polymorphisms in Rv1339 and resistant mutants isolated from the third compound had single nucleotide polymorphisms in QcrB, the proposed target for the series. All the strains were resistant to two compounds, regardless of the mutation, and a strain carrying the QcrB T313I mutation was resistant to all of the imidazopyridine derivatives tested, confirming cross-resistance. By monitoring pH homeostasis and ATP generation, we confirmed that compounds from the series were targeting QcrB; imidazopyridines disrupted pH homeostasis and depleted ATP, providing further evidence of an effect on the electron transport chain. A representative compound was bacteriostatic against replicating bacteria, consistent with a mode of action against QcrB. The series had a narrow inhibitory spectrum, with no activity against other bacterial species. No synergy or antagonism was seen with other antituberculosis drugs under development. In conclusion, our data support the hypothesis that the imidazopyridine series functions by reducing ATP generation via inhibition of QcrB.

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB.

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure-activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.

The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents.

We identified a di-substituted triazolopyrimidine with anti-tubercular activity against Mycobacterium tuberculosis. Three segments of the scaffold were examined rationally to establish a structure-activity relationship with the goal of improving potency and maintaining good physicochemical properties. A number of compounds displayed sub-micromolar activity against Mycobacterium tuberculosis with no cytotoxicity against eukaryotic cells. Non-substituted aromatic rings at C5 and a two-carbon chain connecting a terminal aromatic at C7 were preferred features; the presence of NH at C7 and a lack of substituent at C2 were essential for potency. We identified compounds with acceptable metabolic stability in rodent and human liver microsomes. Our findings suggest that the easily-synthesized triazolopyrimidines are a promising class of potent anti-tubercular agents and warrant further investigation in our search for new drugs to fight tuberculosis.

In Vitro Evaluation of Novel Nitazoxanide Derivatives against Mycobacterium tuberculosis.

Nitazoxanide has antiparasitic and antibiotic activities including activity against Mycobacterium tuberculosis. We prepared and evaluated a set of its analogues to determine the structure-activity relationship, and identified several amide- and urea-based analogues with low micromolar activity against M. tuberculosis in vitro. Pharmacokinetics in the rat suggested a path forward to obtain bioavailable compounds. The series had a good microbiological profile with bactericidal activity in vitro against replicating and nonreplicating M. tuberculosis. Analogues had limited activity against other Gram-positive bacteria but no activity against Gram-negative bacteria. Our studies identified the key liability in this series as cytotoxicity. Future work concentrating on identifying the target(s) could assist in removing activity against eukaryotic cells.

Synthesis and Evaluation of the 2-Aminothiazoles as Anti-Tubercular Agents.

The 2-aminothiazole series has anti-bacterial activity against the important global pathogen Mycobacterium tuberculosis. We explored the nature of the activity by designing and synthesizing a large number of analogs and testing these for activity against M. tuberculosis, as well as eukaryotic cells. We determined that the C-2 position of the thiazole can accommodate a range of lipophilic substitutions, while both the C-4 position and the thiazole core are sensitive to change. The series has good activity against M. tuberculosis growth with sub-micromolar minimum inhibitory concentrations being achieved. A representative analog was selective for mycobacterial species over other bacteria and was rapidly bactericidal against replicating M. tuberculosis. The mode of action does not appear to involve iron chelation. We conclude that this series has potential for further development as novel anti-tubercular agents.

Identification of Phenoxyalkylbenzimidazoles with Antitubercular Activity.

We conducted an evaluation of the phenoxyalkylbenzimidazole series based on the exemplar 2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole for its antitubercular activity. Four segments of the molecule were examined systematically to define a structure-activity relationship with respect to biological activity. Compounds had submicromolar activity against Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration (MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523). Compounds were selective for M. tuberculosis over other bacterial species, including the closely related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobically grown, replicating M. tuberculosis, but were bactericidal against nonreplicating bacteria. Representative compounds had moderate to high permeability in MDCK cells, but were rapidly metabolized in rodents and human liver microsomes, suggesting the possibility of rapid in vivo hepatic clearance mediated by oxidative metabolism. These results indicate that the readily synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antitubercular agents, if the metabolic liability can be solved.

Discovery of 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a pan-RAF inhibitor with minimal paradoxical activation and activity against BRAF or RAS mutant tumor cells.

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

Synthesis and evaluation of the 2,4-diaminoquinazoline series as anti-tubercular agents.

The 2,4-diaminoquinazoline class of compounds has previously been identified as an effective inhibitor of Mycobacterium tuberculosis growth. We conducted an extensive evaluation of the series for its potential as a lead candidate for tuberculosis drug discovery. Three segments of the representative molecule N-(4-fluorobenzyl)-2-(piperidin-1-yl)quinazolin-4-amine were examined systematically to explore structure-activity relationships influencing potency. We determined that the benzylic amine at the 4-position, the piperidine at 2-position and the N-1 (but not N-3) are key activity determinants. The 3-deaza analog retained similar activity to the parent molecule. Biological activity was not dependent on iron or carbon source availability. We demonstrated through pharmacokinetic studies in rats that good in vivo compound exposure is achievable. A representative compound demonstrated bactericidal activity against both replicating and non-replicating M. tuberculosis. We isolated and sequenced M. tuberculosis mutants resistant to this compound and observed mutations in Rv3161c, a gene predicted to encode a dioxygenase, suggesting that the compound may act as a pro-drug.

A high-throughput screen against pantothenate synthetase (PanC) identifies 3-biphenyl-4-cyanopyrrole-2-carboxylic acids as a new class of inhibitor with activity against Mycobacterium tuberculosis.

The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.

Lipoamide channel-binding sulfonamides selectively inhibit mycobacterial lipoamide dehydrogenase.

Tuberculosis remains a global health emergency that calls for treatment regimens directed at new targets. Here we explored lipoamide dehydrogenase (Lpd), a metabolic and detoxifying enzyme in Mycobacterium tuberculosis (Mtb) whose deletion drastically impairs Mtb's ability to establish infection in the mouse. Upon screening more than 1.6 million compounds, we identified N-methylpyridine 3-sulfonamides as potent and species-selective inhibitors of Mtb Lpd affording >1000-fold selectivity versus the human homologue. The sulfonamides demonstrated low nanomolar affinity and bound at the lipoamide channel in an Lpd-inhibitor cocrystal. Their selectivity could be attributed, at least partially, to hydrogen bonding of the sulfonamide amide oxygen with the species variant Arg93 in the lipoamide channel. Although potent and selective, the sulfonamides did not enter mycobacteria, as determined by their inability to accumulate in Mtb to effective levels or to produce changes in intracellular metabolites. This work demonstrates that high potency and selectivity can be achieved at the lipoamide-binding site of Mtb Lpd, a site different from the NAD⁺/NADH pocket targeted by previously reported species-selective triazaspirodimethoxybenzoyl inhibitors.

Advancement of Imidazo[1,2-a]pyridines with Improved Pharmacokinetics and Nanomolar Activity Against Mycobacterium tuberculosis.

A set of fourteen imidazo[1,2-a]pyridine-3-carboxamides was synthesized and screened against Mycobacterium tuberculosis H37Rv. The minimum inhibitory concentrations of twelve of these agents were ≤ 1 µM against replicating bacteria and five compounds (9, 12, 16, 17 and 18) had MIC values ≤ 0.006 µM. Compounds 13 and 18 were screened against a panel of MDR and XDR drug resistant clinical Mtb strains with the potency of 18 surpassing that of clinical candidate PA-824 by nearly 10 fold. The in vivo pharmacokinetics of compounds 13 and 18 were evaluated in male mice by oral (PO) and intravenous (IV) routes. These results indicate that readily synthesized imidazo[1,2-a]pyridine-3-carboxamides are an exciting new class of potent, selective anti-TB agents that merit additional development opportunities.

Advent of Imidazo[1,2-a]pyridine-3-carboxamides with Potent Multi- and Extended Drug Resistant Antituberculosis Activity.

A set of nine 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides and one 2,6-dimethylimidazo[1,2-a]pyrimidine-3-carboxamide were synthesized. The compounds were evaluated for their in vitro anti-tuberculosis activity versus replicating, non-replicating, multi- and extensive drug resistant Mtb strains. The MIC(90) values of seven of these agents were ≤ 1 µM against the various tuberculosis strains tested. A representative compound of this class (1) was screened against seven non-tubercular strains as well as other non-mycobacteria organisms and demonstrated remarkable microbe selectivity. A transcriptional profiling experiment of Mtb treated with compound 1 was performed to give a preliminary indication of the mode of action. Lastly, the in vivo ADME properties of compounds 1, 3, 4, and 6 were assessed. The 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides are a drug-like and synthetically accessible class of anti-TB agents that have excellent selective potency against multi- and extensive drug resistant TB and encouraging pharmacokinetics.