Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB-DnaK bichaperone system.

The ClpB-DnaK bichaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an antitumor target. We discovered that N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a Kd∼60 µM and inhibits the casein-activated, but not the basal, ATPase activity of ClpB with an IC50∼5 µM. The remaining p97 ligands, which displayed a higher affinity toward p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a Kd∼100 µM and did not affect the DnaK ATPase but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB-DnaK bichaperone system in vitro with an IC50∼5 µM and suppressed the growth of cultured Escherichia coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ toward ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.

Revisiting the ß-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

The suboptimal effectiveness of ß-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a ß-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of M. tuberculosis to ß-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of ß-lactams and that small-molecule inhibitors of those accessory targets might sensitize M. tuberculosis to ß-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize M. tuberculosis to meropenem. We identified six hit compounds, belonging to either the N-arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the N-arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the N-arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by ß-lactam accessory proteins. ß-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

UNLABELLED: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Threading the Needle: Small-Molecule Targeting of a Xenobiotic Receptor to Ablate Escherichia coli Polysaccharide Capsule Expression Without Altering Antibiotic Resistance.

BACKGROUND: Uropathogenic Escherichia coli (UPEC), a leading cause of urinary tract and invasive infections worldwide, is rapidly acquiring multidrug resistance, hastening the need for selective new anti-infective agents. Here we demonstrate the molecular target of DU011, our previously discovered potent, nontoxic, small-molecule inhibitor of UPEC polysaccharide capsule biogenesis and virulence. METHODS: Real-time polymerase chain reaction analysis and a target-overexpression drug-suppressor screen were used to localize the putative inhibitor target. A thermal shift assay quantified interactions between the target protein and the inhibitor, and a novel DNase protection assay measured chemical inhibition of protein-DNA interactions. Virulence of a regulatory target mutant was assessed in a murine sepsis model. RESULTS: MprA, a MarR family transcriptional repressor, was identified as the putative target of the DU011 inhibitor. Thermal shift measurements indicated the formation of a stable DU011-MprA complex, and DU011 abrogated MprA binding to its DNA promoter site. Knockout of mprA had effects similar to that of DU011 treatment of wild-type bacteria: a loss of encapsulation and complete attenuation in a murine sepsis model, without any negative change in antibiotic resistance. CONCLUSIONS: MprA regulates UPEC polysaccharide encapsulation, is essential for UPEC virulence, and can be targeted without inducing antibiotic resistance.

Potency enhancement of the κ-opioid receptor antagonist probe ML140 through sulfonamide constraint utilizing a tetrahydroisoquinoline motif.

Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined with additional structure-activity relationship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.

Primuline derivatives that mimic RNA to stimulate hepatitis C virus NS3 helicase-catalyzed ATP hydrolysis.

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 µM. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays.

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50=1.4 µM), suramin sodium salt (IC50=3.6 µM), NF 023 hydrate (IC50=6.2 µM) and tyrphostin AG 538 (IC50=3.6 µM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

Skeletal diversification via heteroatom linkage control: preparation of bicyclic and spirocyclic scaffolds from N-substituted homopropargyl alcohols.

The discovery and application of a new branching pathway synthesis strategy that rapidly produces skeletally diverse scaffolds is described. Two different scaffold types, one a bicyclic iodo-vinylidene tertiary amine/tertiary alcohol and the other, a spirocyclic 3-furanone, are each obtained using a two-step sequence featuring a common first step. Both scaffold types lead to intermediates that can be orthogonally diversified using the same final components. One of the scaffold types was obtained in sufficiently high yield that it was immediately used to produce a 97-compound library.

Benzylmorpholine analogs as selective inhibitors of lung cytochrome P450 2A13 for the chemoprevention of lung cancer in tobacco users.

PURPOSE: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. METHODS: Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design. RESULTS: A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. CONCLUSIONS: Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.

Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: development of chemical probe ML315.

Substituted pyrimidine inhibitors of the Clk and Dyrk kinases have been developed, exploring structure-activity relationships around four different chemotypes. The most potent compounds have low-nanomolar inhibitory activity against Clk1, Clk2, Clk4, Dyrk1A and Dyrk1B. Kinome scans with 442 kinases using agents representing three of the chemotypes show these inhibitors to be highly selective for the Clk and Dyrk families. Further off-target pharmacological evaluation with ML315, the most selective agent, supports this conclusion.

Novel compound C150 inhibits pancreatic cancer through induction of ER stress and proteosome assembly.

Pancreatic cancer is a devastating disease with a dismal prognosis and poor treatment outcomes. Searching for new agents for pancreatic cancer treatment is of great significance. We previously identified a novel activity of compound C150 to inhibit pancreatic cancer epithelial-to-mesenchymal transition (EMT). Here, we further revealed its mechanism of action. C150 induced ER stress in pancreatic cancer cells and subsequently increased proteasome activity by enhancing proteasome assembly, which subsequently enhanced the degradation of critical EMT transcription factors (EMT-TFs). In addition, as cellular responses to ER stress, autophagy was elevated, and general protein synthesis was inhibited in pancreatic cancer cells. Besides EMT inhibition, the C150-induced ER stress resulted in G2/M cell cycle arrest, which halted cell proliferation and led to cellular senescence. In an orthotopic syngeneic mouse model, an oral dose of C150 at 150 mg/kg 3× weekly significantly increased survival of mice bearing pancreatic tumors, and reduced tumor growth and ascites occurrence. These results suggested that compound C150 holds promises in comprehensively inhibiting pancreatic cancer progression.

DNAJA1- and conformational mutant p53-dependent inhibition of cancer cell migration by a novel compound identified through a virtual screen.

Cancers are frequently addicted to oncogenic missense mutant p53 (mutp53). DNAJA1, a member of heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), plays a crucial role in the stabilization and oncogenic activity of misfolded or conformational mutp53 by binding to and preventing mutp53 from proteasomal degradation. However, strategies to deplete mutp53 are not well-established, and no HSP40/JDPs inhibitors are clinically available. To identify compounds that bind to DNAJA1 and induce mutp53 degradation, we performed an in silico docking study of ~10 million of compounds from the ZINC database for the J-domain of DNAJA1. A compound 7-3 was identified, and its analogue A11 effectively reduced the levels of DNAJA1 and conformational mutp53 with minimal effects on the levels of wild-type p53 and DNA-contact mutp53. A11 suppressed migration and filopodia formation in a manner dependent on DNAJA1 and conformational mutp53. A mutant DNAJA1 with alanine mutations at predicted amino acids (tyrosine 7, lysine 44, and glutamine 47) failed to bind to A11. Cells expressing the mutant DNAJA1 became insensitive to A11-mediated depletion of DNAJA1 and mutp53 as well as A11-mediated inhibition of cell migration. Thus, A11 is the first HSP40/JDP inhibitor that has not been previously characterized for depleting DNAJA1 and subsequently conformational mutp53, leading to inhibition of cancer cell migration. A11 can be exploited for a novel treatment against cancers expressing conformational mutp53.

Discovery and Optimization of Pyrrolopyrimidine Derivatives as Selective Disruptors of the Perinucleolar Compartment, a Marker of Tumor Progression toward Metastasis.

The perinucleolar compartment (PNC) is a dynamic subnuclear body found at the periphery of the nucleolus. The PNC is enriched with RNA transcripts and RNA-binding proteins, reflecting different states of genome organization. PNC prevalence positively correlates with cancer progression and metastatic capacity, making it a useful marker for metastatic cancer progression. A high-throughput, high-content assay was developed to identify novel small molecules that selectively reduce PNC prevalence in cancer cells. We identified and further optimized a pyrrolopyrimidine series able to reduce PNC prevalence in PC3M cancer cells at submicromolar concentrations without affecting cell viability. Structure-activity relationship exploration of the structural elements necessary for activity resulted in the discovery of several potent compounds. Analysis of in vitro drug-like properties led to the discovery of the bioavailable analogue, metarrestin, which has shown potent antimetastatic activity with improved survival in rodent models and is currently being evaluated in a first-in-human phase 1 clinical trial.

Novel Compound C150 Inhibits Pancreatic Cancer Cell Epithelial-to-Mesenchymal Transition and Tumor Growth in Mice.

Pancreatic cancer cell epithelial-to-mesenchymal transition (EMT) is an important contributor to cell invasion and tumor progression. Therefore, targeting EMT may be beneficial for pancreatic cancer treatment. The aim of the present study was to report on the inhibitory effect of the novel compound C150 on the EMT of pancreatic cancer cells. C150 inhibited cell proliferation in multiple pancreatic cancer cells with IC50 values of 1-2.5 µM, while in an non-cancerous pancreatic epithelial cell line hTERT-HPNE the IC50 value was >12.5 µM. C150 significantly inhibited pancreatic cancer cell migration and invasion, as demonstrated by 3-dimensional cell invasion, wound healing and Boyden chamber Transwell migration-invasion assays. Moreover, C150 treatment decreased MMP-2 gene expression in PANC-1 cells and reduced MMP-2 activity in gelatin zymography assay. In an orthotopic mouse model of pancreatic cancer, C150 significantly reduced tumor growth at the dose of 15 mg/kg by intraperitoneal injection three times per week. Furthermore, C150 enhanced protein degradation of Snail, an important EMT-promoting transcription factor, and decreased the expression of the mesenchymal marker N-cadherin, while it increased the expression of the epithelial markers zonula occludens-1 and claudin-1. The findings of the present study suggested that C150 is a novel EMT inhibitor that may be promising for inhibiting pancreatic cancer growth and metastasis.

Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases.

As ß-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb's survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these ß-lactams to target diverse classes of proteins.

Identification of Antimalarial Inhibitors Using Late-Stage Gametocytes in a Phenotypic Live/Dead Assay.

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at risk of contracting malaria and an estimated 450,000 deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector. This stage is a bottleneck for reproduction of the parasite, making it a target for small-molecule drug discovery. Targeting this stage, we used whole Plasmodium falciparum gametocytes from in vitro culture and implemented them into 1536-well plates to create a live/dead phenotypic antigametocyte assay. Using specialized equipment and upon further validation, we screened ~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified 100 primary screening hits that were tested for concentration response. Additional follow-up studies to determine specificity, potency, and increased efficacy of the antigametocyte candidate compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13 chemical analogs were subsequently tested as de novo powders, which confirmed original activity from the initial analysis and now provide a point of future engagement.

Effect of C-2 substitution on the stability of non-traditional cephalosporins in mouse plasma.

A systematic study of the stability of a set of cephalosporins in mouse plasma reveals that cephalosporins lacking an acidic moiety at C-2 may be vulnerable to ß-lactam cleavage in mouse plasma.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.