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Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of all αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab’s yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFβ activation. In IPF patient lung fibroblasts, TGFβ treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated superior in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFβ action though mechanisms beyond the inhibition of latent TGFβ activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.One Sentence Summarytargeting integrin in lung fibrosis

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

Multiple integrins have been implicated in modulating renal function. Modulation of integrin function can lead to pathophysiological processes associated with diabetic nephropathy such as alterations in the glomerular filtration barrier and kidney fibrosis. The complexity of these pathophysiological changes implies that multiple integrin subtypes might need to be targeted to ameliorate the progression of renal disease. To address this hypothesis, we investigated the effects of MK‐0429, a compound that was originally developed as an αvβ3 inhibitor for the treatment of osteoporosis, on renal function and fibrosis. We demonstrated that MK‐0429 is an equipotent pan‐inhibitor of multiple av integrins. MK‐0429 dose‐dependently inhibited podocyte motility and also suppressed TGF‐β‐induced fibrosis marker gene expression in kidney fibroblasts. Moreover, in the obese ZSF1 rat model of diabetic nephropathy, chronic treatment with MK‐0429 resulted in significant reduction in proteinuria, kidney fibrosis, and collagen accumulation. In summary, our results suggest that inhibition of multiple integrin subtypes might lead to meaningful impact on proteinuria and renal fibrosis in diabetic nephropathy.

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure–activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.

Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure–activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure–activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.

Efforts were dedicated to develop potent and brain penetrant prolylcarboxypeptidase (PrCP) inhibitors by replacing the amide group of original leads 1 and 2 with heterocycles. Aminopyrimidines including compound 32a were identified to display good PrCP inhibitory activity (32a, IC(50)=43 nM) and impressive ability to penetrate brain in mice (brain/plasma ratio: 1.4).

The generation of peptides presented by MHC class I molecules requires the proteolytic activity of the proteasome and/or other peptidases. The processing of a short vaccinia virus-encoded antigen can take place by a proteasome-independent pathway involving initiator caspase-5 and -10, which generate antigenic peptides recognized by CD8(+) T lymphocytes. In the present study, comparing single versus double enzyme digestions by mass spectrometry analysis, both qualitative and quantitative differences in the products obtained were identified. These in vitro data suggest that each enzyme can use the degradation products of the other as substrate for new cleavages, indicating concerted endoproteolytic activity of caspase-5 and -10., This work was supported by grants provided by the European Union, Ministerio de Ciencia e Innovación, Comunidad de Madrid, and Instituto de Salud Carlos III to M. D. V., and by Programa Ramón y Cajal and FIPSE Foundation to D. L.

A series of benzimidazole pyrrolidinyl amides containing a piperidinyl group were discovered as novel prolylcarboxypeptidase (PrCP) inhibitors. Low-nanomolar IC(50)'s were achieved for several analogs, of which compound 9b displayed modest ex vivo target engagement in eDIO mouse plasma. Compound 9b was also studied in vivo for its effect on weight loss and food intake in an eDIO mouse model and the results will be discussed.

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

A series of spiroimidazolidinone NPC1L1 inhibitors was discovered by virtual screening of the Merck corporate sample repository using 3D-similarity-based screening. Selection of 330 compounds for testing in an in vitro NPC1L1 binding assay yielded six hits in six distinct chemical series. Follow-up 2D similarity searching yielded several sub- to low-micromolar leads; among these was spiroimidazolidinone 10, with an IC(50) of 2.5 microM. Compound 10 provided a useful scaffold to initiate a medicinal chemistry campaign.

Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.

Mass spectrometry (MS)-based methods coupled to reverse phase chromatography separation are a useful technology to analyze complex peptide pools that are comprised of different peptides with unrelated sequences. In antigen presentation, proteasomes generate a set of short peptides that are closely related and overlapping and in some instances may even have identical retention times and identical masses. In these situations, micro-liquid chromatography-MS/MS focused on each theoretical parent ion followed by manual interpretation optimizes the identification of generated peptides. The results suggest that the degradation of short antigens by the proteasome occurs by sequential cleavage. This work was supported by grants from the Programa Ramón y Cajal, Comunidad de Madrid, Instituto de Salud Carlos III, Fundación FIPSE, and Fondo de Investigaciones Sanitarias de la SS (to D. L.) and from the Ministerio de Educación y Ciencia, Comunidad de Madrid, and Instituto de Salud Carlos III (to M. D. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sí

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.

Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure–activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.

Background: Granzyme B, one of the most abundant granzymes in cytotoxic T-lymphocyte (CTL) granules, and members of the caspase (cysteine aspartyl proteinases) family have a unique cleavage specificity for aspartic acid in P1 and play critical roles in the biochemical events that culminate in cell death. Results: We have determined the three-dimensional structure of the complex of the human granzyme B with a potent tetrapeptide aldehyde inhibitor. The Asp-specific S1 subsite of human granzyme B is significantly larger and less charged than the corresponding Asp-specific site in the apoptosis-promoting caspases, and also larger than the corresponding subsite in rat granzyme B. Conclusions: The above differences account for the variation in substrate specificity among granzyme B, other serine proteases and the caspases, and enable the design of specific inhibitors that can probe the physiological functions of these proteins and the disease states with which they are associated.

Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.

Novel potent and selective mineralocorticoid receptor antagonists were identified, utilizing heterocyclic amide replacements in the oxazolidinedione series. Structure–activity relationship (SAR) efforts focused on improving lipophilic ligand efficiency (LLE) while maintaining nuclear hormone receptor selectivity and reasonable pharmacokinetic profiles.

In the course of screening for antifungal agents we have discovered a novel compound isolated from an endophytic fungus that inhibits fungal sphingolipid synthesis. Khafrefungin, which is composed of aldonic acid linked via an ester to a C22 modified alkyl chain, has fungicidal activity against Candida albicans,Cryptococcus neoformans, and Saccharomyces cerevisiae. Sphingolipid synthesis is inhibited in these organisms at the step in which phosphoinositol is transferred to ceramide, resulting in accumulation of ceramide and loss of all of the complex sphingolipids. In vitro, khafrefungin inhibits the inositol phosphoceramide synthase of C. albicans with an IC50 of 0.6 nm. Khafrefungin does not inhibit the synthesis of mammalian sphingolipids thus making this the first reported compound that is specific for the fungal pathway.

There is compelling evidence that members of the caspase (interleukin-1beta converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 and Caenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.

Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. Restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

Finasteride is employed in treatment of benign prostatic hyperplasia in man, where its target enzyme is steroid 5α-reductase. It is a novel, potent mechanism-based inhibitor of the human prostate (type 2) isozyme. Although it is accepted as an alternate substrate and is ultimately reduced to dihydrofinasteride, this proceeds through an enzyme-bound NADP−dihydrofinasteride adduct. Finasteride is processed with a second-order rate constant, ki/Ki = 1 × 106 M-1 s-1, that approaches kcat/Km for reduction of testosterone, 3 × 106 M-1 s-1, and essentially every catalytic event is lethal (partition ratio ≤ 1.07). The membrane-bound enzyme−inhibitor complex formed from [3H]finasteride appears to release [3H]dihydrofinasteride with a half-life of 1 month at 37 °C (k = (2.57 ± 0.03) × 10-7 s-1), as identified by mass spectroscopy. The intermediate NADP−dihydrofinasteride adduct can be recovered intact by denaturation of the enzyme−inhibitor complex and has been purified. Free in solution, it likewise decomposes to ...

The charybdotoxin (ChTX) receptor has been purified from bovine aortic smooth muscle using conventional chromatographic techniques and sucrose gradient centrifugation. Fractions from the final sucrose gradient purification were enriched in specific binding of monoiodinated ChTX (125i-ChTX) approximately 2000-fold over native sarcolemmal membranes. The ChTX binding activity correlated with the presence of two polypeptides of 65 (alpha) and 31 (beta) kDa. Using the cross-linking reagent, disuccinimidyl suberate, 125I-ChTX was specifically incorporated into a polypeptide of approximately 31 kDa. Cross-linking and binding of 125I-ChTX to the purified ChTX receptor was inhibited by ChTX, iberiotoxin (IbTX), and tetraethylammonium (TEA). Liposomes containing the purified ChTX receptor were incorporated into planar lipid bilayers. In symmetric 150 mM KC1, the channels observed were > 20-fold more selective for potassium over sodium and exhibited a large, single-channel conductance of 323 +/- 2.5 pS in charged lipids and 249 +/- 7 pS in neutral lipids. Depolarizing membrane potentials increased the open probability of the purified channels e-fold per 11.5 +/- 0.3 mV, while intracellular calcium increased the open probability according to a third power (2.9 +/- 0.2) relationship. Mean channel closed durations decreased while open times slightly increased as membrane potential and calcium concentration were elevated. The distributions of open and closed durations were well described by the sums of three and five to six exponential components, respectively. Purified maxi-K channels were blocked with micromolar affinity by external TEA and with nanomolar affinity by extracellular IbTX and ChTX. Kinetics of ChTX block of the purified channel revealed an equilibrium dissociation constant for toxin block 4.6 +/- 0.7 nM under conditions of physiological ionic strength. The purified maxi-K channel displays many of the biophysical and pharmacological properties of maxi-K channels derived from native tissue.

In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11β-HSD1 inhibitors, we examined a set of 11β-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11β-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11β-HSD1 independent pathway.

A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC50 values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.

A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert -butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC 50 values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.

Three new toxins from the venom of the scorpion Leiurus quinquestriatus var. hebraeus have been identified on the basis of their ability to block the Shaker K+ channel. These toxins have been purified using HPLC techniques and characterized as 38 amino acid peptides by mass spectroscopy, amino acid analysis, and sequence determination. Their chemical identity was confirmed by producing fully functional synthetic toxins using recombinant methods. These peptides are potent inhibitors of the Shaker K+ channel (Kd < 1 nM) as well as the mammalian homologues of Shaker. They are related to other previously described K+ channel toxins, but form a new subclass within the larger family of K+ channel inhibitors derived from scorpion venom. We have named these toxins agitoxin 1, 2, and 3, respectively.

The charybdotoxin receptor, purified from bovine tracheal smooth muscle, consists of two subunits (alpha and beta) and, when reconstituted into planar lipid bilayers, forms functional high conductance Ca(2+)-activated K+ channels. Amino acid sequence, obtained from proteolytic fragments of the beta-subunit, was used to design oligonucleotide probes with which cDNAs encoding this protein were isolated. The cDNAs encode a protein of 191 amino acids that contains two hydrophobic (putative transmembrane) domains and bears little sequence homology to subunits of other known ion channels. Site-directed antisera, raised against putative extracellular epitopes of this protein, specifically immunoprecipitated 125I-labeled Bolton-Hunter beta-subunit as well as [125I]charybdotoxin-cross-linked beta-subunit. Under nondenaturing conditions, however, these anti-beta sera immunoprecipitated a complex consisting of both the alpha- and beta-subunits. The data demonstrate that, in vivo, the high conductance Ca(2+)-activated K+ channel exists as a multimer containing both alpha- and beta-subunits, and this cDNA represents the first beta-subunit of a potassium channel cloned to date. Furthermore, we demonstrate that the cloned protein is the subunit to which charybdotoxin is specifically and covalently incorporated when cross-linked to the channel.

Target inactivation analysis was used to determine the functional size of the charybdotoxin (ChTX) receptor in aortic and tracheal sarcolemmal membrane vesicles. This receptor has previously been shown to be an integral component of the high-conductance Ca2+-activated K+ (Maxi-K) channel in these smooth muscles. Exposure of either bovine aortic or bovine tracheal sarcolemma to high-energy irradiation results in disappearance of 125I-labeled ChTX binding activity as a monoexponential function of radiation dose; from these functions molecular masses of 88 +/- 10 kDa and 89 +/- 6 kDa, respectively, can be calculated. Similar results were obtained from radiation inactivation studies with the detergent-solubilized ChTX receptor from aortic sarcolemmal membranes. The effect of radiation on 125I-labeled ChTX binding is to decrease the number of functional ChTX receptors without affecting the affinity of receptors for the toxin, indicating that radiation is destroying, rather than altering, the binding site. The validity of the radiation inactivation technique in these membrane preparations is supported by data obtained in parallel experiments in which target sizes of the alpha 1 subunit of the L-type Ca2+ channel and 5'-nucleotidase were measured. The molecular masses determined for these entities are in excellent agreement with those expected from previous studies. The present data are discussed in terms of the recently determined subunit composition of the smooth muscle Maxi-K channel. In light of the target size, a single alpha beta subunit heterodimer complex could serve as the ChTX receptor.

Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block.

High conductance Ca(2+)-activated K+ (maxi-K) channels from bovine tracheal and aortic smooth muscle membranes have been purified employing monoiodotyrosine charybdotoxin binding as a marker for the channel and conventional chromatographic techniques. This K+ channel is composed of two subunits, alpha and beta, of 62 and 31 kDa, respectively. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electroeluted tracheal smooth muscle alpha-subunit was subjected to tryptic cleavage and a number of fragments were isolated by microbore C18 high performance liquid chromatography. Several of these peptides were microsequenced using Edman degradation techniques. Amino acid sequence information obtained from these fragments reveals the existence of very high sequence homology with the recently cloned mSlo maxi-K channel (Butler, A., Tsunoda, S., McCobb, D. P., Wei, A., and Salkoff, L. (1993) Science 261, 221-224). A specific anti-peptide antibody directed against the amino acid sequence of one of the fragments of the alpha-subunit is capable of specifically immunoprecipitating not only the denatured 125I-Bolton-Hunter-labeled alpha-subunit, but also, under nondenaturing conditions, the complex of alpha and beta subunits, demonstrating specific noncovalent association of both subunits. Thus, our results indicate that the alpha-subunit of the purified tracheal smooth muscle maxi-K channel is a member of the mSlo family of K+ channels and forms a noncovalent complex with a beta-subunit. It is concluded that the extensive biochemical information acquired to date on smooth muscle charybdotoxin receptors is pertinent to the structure of native maxi-K channels.

The high-conductance Ca(2+)-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent homogeneity by a combination of conventional chromatographic techniques and sucrose density gradient centrifugation. Fractions with the highest specific activity for binding of monoiodotyrosine charybdotoxin, [125I]ChTX, were enriched approximately 2000-fold over the initial digitonin-solubilized material up to a specific activity of 1 nmol/mg protein. Silver staining after SDS-polyacrylamide gel electrophoresis of the fractions from the last step of the purification indicates that binding activity is correlated with a major component of the preparation that displays an apparent molecular weight of 62,000. Labeling the same preparation with 125I-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent stoichiometry of 1:1, comigrating with binding activity. The beta subunit is heavily glycosylated. Deglycosylation studies indicate that the beta subunit represents the protein to which [125I]ChTX is covalently incorporated in the presence of the bifunctional cross-linking reagent disuccinimidyl suberate. Binding of [125I]ChTX to the purified ChTX receptor displayed the same pharmacological profile that has been found previously for toxin binding to native membranes, including inhibition by iberiotoxin, limbatustoxin, tetraethylamonium, potassium, cesium, and barium. The purified preparation was reconstituted into liposomes which were then fused with artificial lipid bilayers. Single channels were readily observed with a conductance of 235 picosiemens in 150 mM KCl that displayed selectivity for potassium over chloride and that were blocked by ChTX. The open probability of these channels was increased by depolarizing membrane potentials and by raising the internal calcium concentration. These data suggest that the maxi-K channel purified from tracheal smooth muscle is composed of two subunits.

Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarboxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.

Novel prolylcarboxypeptidase (PrCP) inhibitors with nanomolar IC50 values were prepared by replacing the previously described dichlorobenzimidazole-substituted pyrrolidine amides with a variety of substituted benzylamine amides. In contrast to prior series, the compounds demonstrated minimal inhibition shift in whole serum and minimal recognition by P-glycoprotein (P-gp) efflux transporters. The compounds were also cell permeable and demonstrated in vivo brain exposure. The in vivo effect of compound (S)-6e on weight loss in an established diet-induced obesity (eDIO) mouse model was studied.

A novel peptidyl inhibitor of K+ channels has been purified to homogeneity from venom of the new world scorpion Centruroides margaritatus. The primary structure of this 39-amino-acid peptide, which we term margatoxin (MgTX), was determined by amino acid compositional analysis and peptide sequencing. Margatoxin potently inhibits binding of radiolabeled charybdotoxin (ChTX) to voltage-activated channels in brain synaptic plasma membranes. Like ChTX, MgTX blocks the n-type current of human T-lymphocytes (Kv1.3 channel), but compared to ChTX, is 20-fold more potent (half-block at approximately 50 pM), has a slower dissociation rate, and has no effect on calcium-activated channels. To demonstrate that these characteristics are due solely to the purified toxin, recombinant MgTX was expressed in Escherichia coli as part of a fusion protein. After cleavage and folding, purified recombinant MgTX displayed the same properties as native peptide. Replacement of the COOH-terminal histidine residue of MgTX with asparagine resulted in a peptide with a 10-fold reduction in potency. This was due to a faster apparent dissociation rate, suggesting that the COOH-terminal amino acid may play an important role in the binding of MgTX to the Kv1.3 channel. MgTX displays significant sequence homology with previously identified K+ channel inhibitors (e.g. ChTX, iberiotoxin, noxiustoxin, and kaliotoxin). However, given its potency and unique selectivity, MgTX represents an especially useful tool with which to study the physiologic role of Kv1.3 channels.

High-conductance Ca2+-activated K+ channels (maxi-K channels) represent a family of proteins that display high selectivity and conductance for K+. With the discovery and purification of selective, high affinity, peptidyl probes for these channels, it has been possible to develop the molecular pharmacology of the maxi-K channel, and address the physiological role which these channels play in target tissues. Although molecular biology approaches have been successful in cloning other types of K+ channels, the molecular constituents of maxi-K channels are unknown. To gain insight into the structural composition of maxi-K channels, the ChTX receptor from bovine aortic and tracheal sarcolemmal membrane vesicles has been solubilized in functional form with digitonin, characterized, and purified to apparent homogeneity. Reconstitution experiments with these preparations demonstrate the presence of maxi-K channel activity with properties that are indistinguishable from those of the native channel. Studies are underway to determine sequence information for the identified ChTX receptor subunits in order to clone and express the maxi-K channel.

Charybdotoxin (ChTX) and iberiotoxin (IbTX) are highly charged peptidyl toxins which exhibit 68% sequence identity and share a similar three-dimensional structure. Despite these structural similarities, IbTX and ChTX differ in their selectivity for two types of potassium channels; large conductance calcium-activated potassium (maxi-K) channels and slowly inactivating voltage-gated (Kv1.3) potassium channels. ChTX blocks with high affinity both maxi-K and Kv1.3 channels, while IbTX blocks the maxi-K but not the voltage-gated channel. To identify regions of the toxins which impart this this selectivity, we have constructed by solid-phase synthesis two chimeric toxins, ChTX1-19IbTX20-37 (Ch-IbTX) and IbTX1-19ChTX20-37 (Ib-ChTX), as well as a truncated peptide, ChTX7-37. These peptides were assayed for their ability to inhibit [125I]ChTX binding in sarcolemmal vesicles from smooth muscle (maxi-K binding) and [125I]ChTX binding to plasma membranes from brain (Kv1.3 binding). The ability of the peptides to block the maxi-K channel was determined from recordings of single maxi-K channels incorporated into planar lipid bilayers. Block of Kv1.3 was determined from recordings of whole cell currents in Xenopus oocytes injected with mRNA encoding the cloned Kv1.3 channel. Both chimeric toxins inhibited [125I]ChTX binding to sarcolemmal membranes from smooth muscle, and they both blocked the maxi-K channel in planar lipid bilayers. In contrast, [125I]ChTX binding in brain and Kv1.3 currents expressed in oocytes were inhibited only by the chimera Ib-ChTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Ezetimibe (Zetia®), a cholesterol-absorption inhibitor (CAI) approved by the FDA for the treatment of hypercholesterolemia, is believed to target the intestine protein Niemann-Pick C1-Like 1 (NPC1L1) or its pathway. A spiroimidazolidinone NPC1L1 inhibitor identified by virtual screening showed moderate binding activity but was not efficacious in an in vivo rodent model of cholesterol absorption. Synthesis of analogs established the structure-activity relationships for binding activity, and resulted in compounds with in vivo efficacy, including 24, which inhibited plasma cholesterol absorption by 67% in the mouse, thereby providing proof-of-concept that non-β-lactams can be effective CAIs.

A novel class of small molecule human granzyme B inhibitors is reported. Compound 20 has a K(i) of 7 nM against human granzyme B and blocks CTL mediated apoptosis with an IC(50) of 3 micromolar.

CD8(+) cytotoxic T lymphocytes recognize infected cells in which MHC class I molecules present pathogen-derived peptides that have been processed mainly by proteasomes. Many infections induce a set of proteases, the caspases involved in apoptosis or inflammation. In this study, we report that processing and presentation of a short vaccinia virus-encoded Ag can take place also by a nonproteasomal pathway, which was blocked in infected cells with chemical inhibitors of caspases. By cleaving at noncanonical sites, at least two caspases generated antigenic peptides recognized by T lymphocytes. The sites and the peptidic products were partially overlapping but different to those used and produced by proteasomes in vitro. Antigenic natural peptides produced in infected cells by either pathway were quantitatively and qualitatively similar. Finally, coexpression of the natural vaccinia virus protein B13, which is an inhibitor of caspases and apoptosis, impaired Ag presentation by the caspase pathway in infected cells. These data support the hypothesis that numerous cellular proteolytic systems, including those induced during infection, such as caspases involved in apoptosis or in inflammation, contribute to the repertoire of presented peptides, thereby facilitating immunosurveillance. This work was supported by grants provided by the European Union, Ministerio de Ciencia e Innovación, Comunidad de Madrid, and Instituto de Salud Carlos III (to M.D.V.) and Programa Ramón y Cajal, Comunidad de Madrid, and Instituto de Salud Carlos III (to D.L.). G.L.S. is a Wellcome Principal Research Fellow. Sí

During our effort to design a receptor binding assay to aid in the elucidation of the molecular mechanism of ezetimibe, we prepared a sulfur-35 containing radioligand which exhibits improved potency over the glucuronide conjugate of ezetimibe in both native enterocyte brush border membranes and membranes from cells expressing recombinant NPC1L1. Herein, we describe the different synthetic strategies which were used to obtain this compound as well as its effectiveness in the aforementioned assay.

Charybdotoxin (ChTX), a 37 amino acid peptide isolated from venom of the scorpion Leiurus quinquesfriafus var, hebraeus, is a potent inhibitor of the high-conductance Ca2+-activated K+ channel (PK,Ca) present in smooth muscle tissues. ChTX has been radiolabeled in biologically active form and its monoiodotyrosine derivative used to study the interaction of toxin with sarcolemmal membrane vesicles derived from different smooth muscles. The receptor site for ChTX in vascular smooth muscle has been solubilized in active form and purified to apparent homogeneity. lberiotoxin (IbTX), purified from Bufhus tamulus scorpion venom, displays high homology in its primary sequence with ChTX. Despite this, lbTX interacts with PK ca at a distinct site and blocks channel activity by a different mechanism than ChTX. Two hybrid peptides, ChTX1-19 lbTX20-37 and IbTX1-19 ChTXzo-37 have been prepared synthetically. Studies with these peptides indicate that the C-terminus part of the hybrid molecule defines the site and mode of interaction of each toxin with PK,Ca, despite significant heterogeneity between the N-termini of both structures. ChTX, lbTX and the hybrid molecules are, therefore, unique tools with which to study the physiological role of PK,c~ in different smooth muscle tissues.

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. K D values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.

A novel class of small molecule human granzyme B inhibitors is reported. Compound 20 has a K(i) of 7 nM against human granzyme B and blocks CTL mediated apoptosis with an IC(50) of 3 micromolar.

Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1 - 10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have kcat/Km values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca2+) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.