Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

A Scalable Platform for Producing Recombinant Nucleosomes with Codified Histone Methyltransferase Substrate Preferences.

Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1; also known as NSD2) is a SET domain-containing histone lysine methyltransferase. A chromosomal translocation occurs in 15-20% of multiple myeloma patients and is associated with increased production of WHSC1 and poor clinical prognosis. To define the substrate requirements of NSD2, we established a platform for the large-scale production of recombinant polynucleosomes, based on authentic human histone proteins, expressed in E. coli, and complexed with linearized DNA. A brief survey of methyltransferases whose substrate requirements are recorded in the literature yielded expected results, lending credence to the fitness of our approach. This platform was readily 'codified' with respect to both position and extent of methylation at histone 3 lysines 18 and 36 and led to the conclusion that the most readily discernible activity of NSD2 in contact with a nucleosome substrate is dimethylation of histone 3 lysine 36. We further explored reaction mechanism, and conclude a processive, rather than distributive mechanism best describes the interaction of NSD2 with intact nucleosome substrates. The methods developed feature scale and flexibility and are suited to thorough pharmaceutical-scale drug discovery campaigns.

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36.

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-(14)C and (36)S and secondary Me-(3)H3, Me-(2)H3, 5'-(14)C, and 5'-(3)H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2.

Nuclear receptor-binding SET domain protein 2 (NSD2) is a histone H3 lysine 36 (H3K36)-specific methyltransferase enzyme that is overexpressed in a number of cancers, including multiple myeloma. NSD2 binds to S-adenosyl-l-methionine (SAM) and nucleosome substrates to catalyze the transfer of a methyl group from SAM to the ε-amino group of histone H3K36. Equilibrium binding isotope effects and density functional theory calculations indicate that the SAM methyl group is sterically constrained in complex with NSD2, and that this steric constraint is released upon nucleosome binding. Together, these results show that nucleosome binding to NSD2 induces a significant change in the chemical environment of enzyme-bound SAM.

GSK356278, a potent, selective, brain-penetrant phosphodiesterase 4 inhibitor that demonstrates anxiolytic and cognition-enhancing effects without inducing side effects in preclinical species.

Small molecule phosphodiesterase (PDE) 4 inhibitors have long been known to show therapeutic benefit in various preclinical models of psychiatric and neurologic diseases because of their ability to elevate cAMP in various cell types of the central nervous system. Despite the registration of the first PDE4 inhibitor, roflumilast, for the treatment of chronic obstructive pulmonary disease, the therapeutic potential of PDE4 inhibitors in neurologic diseases has never been fulfilled in the clinic due to severe dose-limiting side effects such as nausea and vomiting. In this study, we describe the detailed pharmacological characterization of GSK356278 [5-(5-((2,4-dimethylthiazol-5-yl)methyl)-1,3,4-oxadiazol-2-yl)-1-ethyl-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine], a potent, selective, and brain-penetrant PDE4 inhibitor that shows a superior therapeutic index to both rolipram and roflumilast in various preclinical species and has potential for further development in the clinic for the treatment of psychiatric and neurologic diseases. GSK356278 inhibited PDE4B enzyme activity with a pIC50 of 8.8 and bound to the high-affinity rolipram binding site with a pIC50 of 8.6. In preclinical models, the therapeutic index as defined in a rodent lung inflammation model versus rat pica feeding was >150 compared with 0.5 and 6.4 for rolipram and roflumilast, respectively. In a model of anxiety in common marmosets, the therapeutic index for GSK356278 was >10 versus <1 for rolipram. We also demonstrate that GSK356278 enhances performance in a model of executive function in cynomolgus macaques with no adverse effects, a therapeutic profile that supports further evaluation of GSK356278 in a clinical setting.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

The amino-acid substituents of dipeptide substrates of cathepsin C can determine the rate-limiting steps of catalysis.

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k(cat)/K(a) and k(cat) values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k(cat)/K(a) (1.6 ± 0.09 µM(-1) s(-1)) and k(cat) (255 ± 6 s(-1)), an inverse sKIE on k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.6 ± 0.15), a modest, normal sKIE on k(cat) ((D)k(cat) = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s(-1)). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k(ac)) and deacylation (k(dac)) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697-8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k(cat)/K(a) values of 0.49 ± 0.07 µM(-1 )s(-1) and 5.3 ± 0.5 µM(-1 )s(-1), and k(cat) values of 28 ± 1 s(-1) and 25 ± 0.5 s(-1). Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.74 ± 0.05) and normal for k(cat) ((D)k(cat) = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s(-1) and 390 s(-1), respectively), from which it was determined that k(ac) ≫ k(dac) ∼ k(cat). The third substrate type, Gly-Ile-AMC, gave very low values of k(cat)/K(a) (0.0015 ± 0.0001 µM(-1) s(-1)) and k(cat) (0.33 ± 0.02 s(-1)), no sKIEs, ((D)(k(cat)/K(a)) = 1.05 ± 0.5 and (D)k(cat) = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which k(dac) > k(ac) ∼ k(cat), and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P(1) position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases.

A highly selective, cell-permeable furin inhibitor BOS-318 rescues key features of cystic fibrosis airway disease.

In cystic fibrosis (CF), excessive furin activity plays a critical role in the activation of the epithelial sodium channel (ENaC), dysregulation of which contributes to airway dehydration, ineffective mucociliary clearance (MCC), and mucus obstruction. Here, we report a highly selective, cell-permeable furin inhibitor, BOS-318, that derives selectivity by eliciting the formation of a new, unexpected binding pocket independent of the active site catalytic triad. Using human ex vivo models, BOS-318 showed significant suppression of ENaC, which led to enhanced airway hydration and an ∼30-fold increase in MCC rate. Furin inhibition also protected ENaC from subsequent activation by neutrophil elastase, a soluble protease dominant in CF airways. Additional therapeutic benefits include protection against epithelial cell death induced by Pseudomonas aeruginosa exotoxin A. Our findings demonstrate the utility of selective furin inhibition as a mutation-agnostic approach that can correct features of CF airway pathophysiology in a manner expected to deliver therapeutic value.

Methyltransferases prefer monomer over core-trimmed nucleosomes as in vitro substrates.

Epigenetics is an area of increasing interest for drug discovery, driving the need for assays that use nucleosome substrates. Our studies showed that SUV39H1, a histone lysine methyltransferase, and Dnmt3b/Dnmt3L, a DNA methyltransferase, both exhibited approximately five times more activity on monomer nucleosomes than on DNA-core-trimmed nucleosomes in a scintillation proximity assay (SPA). The methyltransferases recognize and have a preference for nucleosomes with longer DNA strands. Our findings suggest that the use of monomer nucleosomes as substrates using SPA technology could lead to more robust screening assays and potentially more specific small molecule inhibitors of epigenetic enzymes.

Kinetic mechanism and rate-limiting steps of focal adhesion kinase-1.

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH(2), FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k(cat) (0.052 +/- 0.001 s(-1)), K(MgATP) (1.2 +/- 0.1 microM), K(iMgATP) (1.3 +/- 0.2 microM), K(FAK-tide) (5.6 +/- 0.4 microM), and K(iFAK-tide) (6.1 +/- 1.1 microM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the betagamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-(18)O(4)]ATP in the presence of 1 mM [gamma-(18)O(4)]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k(x)/k(cat) = 0.14 +/- 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k(cat)/K(FAK-tide), while k(cat) and k(cat)/K(MgATP) were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k(cat)/K(m) for MgATP but not on k(cat)/K(m) for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (k(cat)) is rate-limited by both reversible phosphoryl group transfer (k(forward) approximately 0.2 s(-1) and k(reverse) approximately 0.04 s(-1)) and a slow step (k(conf) approximately 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.

Targeting the Regulatory Site of ER Aminopeptidase 1 Leads to the Discovery of a Natural Product Modulator of Antigen Presentation.

ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.

Identification of PDE4B Over 4D subtype-selective inhibitors revealing an unprecedented binding mode.

A PDE4B over 4D-selective inhibitor programme was initiated to capitalise on the recently discovered predominance of the PDE4B subtype in inflammatory cell regulation. The SAR of a tetrahydrobenzothiophene (THBT) series did not agree with either of two proposed docking modes in the 4B binding site. A subsequent X-ray co-crystal structure determination revealed that the THBT ligand displaces the Gln-443 residue, invariably ligand-anchoring in previous PDE4 co-crystal structures, and even shifts helix-15 by 1-2A. For the first time, several residues of the C-terminus previously proposed to be involved in subtype selectivity are resolved and three of them extend into the ligand binding site potentially allowing for selective drug design.

Chemical mechanism of a cysteine protease, cathepsin C, as revealed by integration of both steady-state and pre-steady-state solvent kinetic isotope effects.

Cathepsin C, or dipeptidyl peptidase I, is a lysosomal cysteine protease of the papain family that catalyzes the sequential removal of dipeptides from the free N-termini of proteins and peptides. Using the dipeptide substrate Ser-Tyr-AMC, cathepsin C was characterized in both steady-state and pre-steady-state kinetic modes. The pH(D) rate profiles for both log k cat/ K m and log k cat conformed to bell-shaped curves for which an inverse solvent kinetic isotope effect (sKIE) of 0.71 +/- 0.14 for (D)( k cat/ K a) and a normal sKIE of 2.76 +/- 0.03 for (D) k cat were obtained. Pre-steady-state kinetics exhibited a single-exponential burst of AMC formation in which the maximal acylation rate ( k ac = 397 +/- 5 s (-1)) was found to be nearly 30-fold greater than the rate-limiting deacylation rate ( k dac = 13.95 +/- 0.013 s (-1)) and turnover number ( k cat = 13.92 +/- 0.001 s (-1)). Analysis of pre-steady-state burst kinetics in D 2O allowed abstraction of a normal sKIE for the acylation half-reaction that was not observed in steady-state kinetics. Since normal sKIEs were obtained for all measurable acylation steps in the presteady state [ (D) k ac = 1.31 +/- 0.04, and the transient kinetic isotope effect at time zero (tKIE (0)) = 2.3 +/- 0.2], the kinetic step(s) contributing to the inverse sKIE of (D)( k cat/ K a) must occur more rapidly than the experimental time frame of the transient kinetics. Results are consistent with a chemical mechanism in which acylation occurs via a two-step process: the thiolate form of Cys-234, which is enriched in D 2O and gives rise to the inverse value of (D)( k cat/ K a), attacks the substrate to form a tetrahedral intermediate that proceeds to form an acyl-enzyme intermediate during a proton transfer step expressing a normal sKIE. The subsequent deacylation half-reaction is rate-limiting, with proton transfers exhibiting normal sKIEs. Through derivation of 12 equations describing all kinetic parameters and sKIEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKIEs allowed the provision of at least one self-consistent set of values for all 13 rate constants in this cysteine protease's chemical mechanism. Simulation of the resulting kinetic profile showed that at steady state approximately 80% of the enzyme exists in an active-site cysteine-acylated form in the mechanistic pathway. The chemical and kinetic details deduced from this work provide a potential roadmap to help steer drug discovery efforts for this and other disease-relevant cysteine proteases.

Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership.

The RecA/LexA axis of the bacterial DNA damage (SOS) response is a promising, yet nontraditional, drug target. The SOS response is initiated upon genotoxic stress, when RecA, a DNA damage sensor, induces LexA, the SOS repressor, to undergo autoproteolysis, thereby derepressing downstream genes that can mediate DNA repair and accelerate mutagenesis. As genetic inhibition of the SOS response sensitizes bacteria to DNA damaging antibiotics and decreases acquired resistance, inhibitors of the RecA/LexA axis could potentiate our current antibiotic arsenal. Compounds targeting RecA, which has many mammalian homologues, have been reported; however, small-molecules targeting LexA autoproteolysis, a reaction unique to the prokaryotic SOS response, have remained elusive. Here, we describe the logistics and accomplishments of an academic-industry partnership formed to pursue inhibitors against the RecA/LexA axis. A novel fluorescence polarization assay reporting on RecA-induced self-cleavage of LexA enabled the screening of 1.8 million compounds. Follow-up studies on select leads show distinct activity patterns in orthogonal assays, including several with activity in cell-based assays reporting on SOS activation. Mechanistic assays demonstrate that we have identified first-in-class small molecules that specifically target the LexA autoproteolysis step in SOS activation. Our efforts establish a realistic example for navigating academic-industry partnerships in pursuit of anti-infective drugs and offer starting points for dedicated lead optimization of SOS inhibitors that could act as adjuvants for current antibiotics.

A simple assay for detection of small-molecule redox activity.

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

Quinoline 3-sulfonamides inhibit lactate dehydrogenase A and reverse aerobic glycolysis in cancer cells.

BACKGROUND: Most normal cells in the presence of oxygen utilize glucose for mitochondrial oxidative phosphorylation. In contrast, many cancer cells rapidly convert glucose to lactate in the cytosol, a process termed aerobic glycolysis. This glycolytic phenotype is enabled by lactate dehydrogenase (LDH), which catalyzes the inter-conversion of pyruvate and lactate. The purpose of this study was to identify and characterize potent and selective inhibitors of LDHA. METHODS: High throughput screening and lead optimization were used to generate inhibitors of LDHA enzymatic activity. Effects of these inhibitors on metabolism were evaluated using cell-based lactate production, oxygen consumption, and 13C NMR spectroscopy assays. Changes in comprehensive metabolic profile, cell proliferation, and apoptosis were assessed upon compound treatment. RESULTS: 3-((3-carbamoyl-7-(3,5-dimethylisoxazol-4-yl)-6-methoxyquinolin-4-yl) amino) benzoic acid was identified as an NADH-competitive LDHA inhibitor. Lead optimization yielded molecules with LDHA inhibitory potencies as low as 2 nM and 10 to 80-fold selectivity over LDHB. Molecules in this family rapidly and profoundly inhibited lactate production rates in multiple cancer cell lines including hepatocellular and breast carcinomas. Consistent with selective inhibition of LDHA, the most sensitive breast cancer cell lines to lactate inhibition in hypoxic conditions were cells with low expression of LDHB. Our inhibitors increased rates of oxygen consumption in hepatocellular carcinoma cells at doses up to 3 microM, while higher concentrations directly inhibited mitochondrial function. Analysis of more than 500 metabolites upon LDHA inhibition in Snu398 cells revealed that intracellular concentrations of glycolysis and citric acid cycle intermediates were increased, consistent with enhanced Krebs cycle activity and blockage of cytosolic glycolysis. Treatment with these compounds also potentiated PKM2 activity and promoted apoptosis in Snu398 cells. CONCLUSIONS: Rapid chemical inhibition of LDHA by these quinoline 3-sulfonamids led to profound metabolic alterations and impaired cell survival in carcinoma cells making it a compelling strategy for treating solid tumors that rely on aerobic glycolysis for survival.

Discovery of novel cyanamide-based inhibitors of cathepsin C.

The discovery of potent and selective cyanamide-based inhibitors of the cysteine protease cathepsin C is detailed. Optimization of the template with regard to plasma stability led to the identification of compound 17, a potent cathepsin C inhibitor with excellent selectivity over other cathepsins and potent in vivo activity in a cigarette smoke mouse model.