Allicin and derivates are cysteine protease inhibitors with antiparasitic activity

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure–activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.     

Structural selectivity of human SGLT inhibitors

Human Na(+)-D-glucose cotransporter (hSGLT) inhibitors constitute the newest class of diabetes drugs, blocking up to 50% of renal glucose reabsorption in vivo. These drugs have potential for widespread use in the diabetes epidemic, but how they work at a molecular level is poorly understood. Here, we use electrophysiological methods to assess how they block Na(+)-D-glucose cotransporter SGLT1 and SGLT2 expressed in human embryonic kidney 293T (HEK-293T) cells and compared them to the classic SGLT inhibitor phlorizin. Dapagliflozin [(1S)-1,5,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucitol], two structural analogs, and the aglycones of phlorizin and dapagliflozin were investigated in detail. Dapagliflozin and fluoro-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-4-F-4-deoxy-D-glucitol] blocked glucose transport and glucose-coupled currents with ≈100-fold specificity for hSGLT2 (K(i) = 6 nM) over hSGLT1 (K(i) = 400 nM). As galactose is a poor substrate for SGLT2, it was surprising that galacto-dapagliflozin [(1S)-1,5-anhydro-1-C-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-galactitol] was a selective inhibitor of hSGLT2, but was less potent than dapagliflozin for both transporters (hSGLT2 K(i) = 25 nM, hSGLT1 K(i) = 25,000 nM). Phlorizin and galacto-dapagliflozin rapidly dissociated from SGLT2 [half-time off rate (t(1/2,Off)) ≈ 20-30 s], while dapagliflozin and fluoro-dapagliflozin dissociated from hSGLT2 at a rate 10-fold slower (t(1/2,Off) ≥ 180 s). Phlorizin was unable to exchange with dapagliflozin bound to hSGLT2. In contrast, dapagliflozin, fluoro-dapagliflozin, and galacto-dapagliflozin dissociated quickly from hSGLT1 (t(1/2,Off) = 1-2 s), and phlorizin readily exchanged with dapagliflozin bound to hSGLT1. The aglycones of phlorizin and dapagliflozin were poor inhibitors of both hSGLT2 and hSGLT1 with K(i) values > 100 μM. These results show that inhibitor binding to SGLTs is composed of two synergistic forces: sugar binding to the glucose site, which is not rigid, and so different sugars will change the orientation of the aglycone in the access vestibule; and the binding of the aglycone affects the binding affinity of the entire inhibitor. Therefore, the pharmacophore must include variations in both the structure of the sugar and the aglycone.     

Measuring and interpreting the selectivity of protein kinase inhibitors

Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.     

Gold compounds as cysteine protease inhibitors: perspectives for pharmaceutical application as antiparasitic agents

Gold compounds form a new class of promising metal-based drugs with a number of potential therapeutic applications, particularly in the fields of anticancer and antimicrobial treatments. Previous research revealed that a group of structurally diverse gold compounds cause conspicuous inhibition of the protease activities of the human proteasome. Given the pharmacological importance of protease inhibition, the present study further explored whether these gold compounds might inhibit a few other proteases that are accepted druggable targets for disease treatment. In particular, four distinct cysteine proteases were considered here: cathepsin B and L that play a primary role in tumor-cell invasion and metastasis; rhodesain, the major cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense and CPB2.8ΔCTE, a Leishmania mexicana mature cysteine protease. Based on the encouraging results obtained for some of the tested gold compounds on the two parasitic cysteine proteases, especially against CPB2.8ΔCTE, with IC_50s in the micromolar range, we next evaluated whether those gold compounds might contrast effectively the growth of the respective protozoa and indeed important antiprotozoal properties were disclosed; on the other hand a certain lack of selectivity was highlighted. Also, no direct or clear correlation could be established between the in vitro antiprotozoal properties and the level of protease inhibition. The implications of these results are discussed in relation to possible pharmaceutical applications.     

Determination of selectivity and efficacy of fatty acid synthesis inhibitors

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.     

Sirtuins inhibitors: The approach to affinity and selectivity

Accumulating evidence has indicated the importance of sirtuins (class III histone deacetylases) in various biological processes. Their potential roles in metabolic and neurodegenerative diseases have encouraged scientists to seek potent and selective sirtuin inhibitors to investigate their biological functions with a view to eventual new therapeutic treatments. This article surveys current knowledge of sirtuin inhibitors including those discovered via high-throughput screening (HST) or via mechanism-based drug design from synthetic or natural sources. Their inhibitory affinity, selectivities, and possible inhibition mechanisms are discussed.     

Benzimidazole-based fXa inhibitors with improved thrombin and trypsin selectivity

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.     

Covalent inhibitors: an opportunity for rational target selectivity

1. Download : Download high-res image (126KB)
2. Download : Download full-size image

     

In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.     

The selectivity of protein kinase inhibitors: a further update

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.     

Differential selectivity of hyaluronidase inhibitors toward acidic and basic hyaluronidases

Hyaluronidase (HAase), a class of enzymes which degrade hyaluronic acid (HA), are involved in the spread of infections/toxins, ovum fertilization, and cancer progression. Thus, HAase inhibitors may have use in disease treatments. We evaluated 21 HAase inhibitors against HYAL-1, testicular, honeybee, and Streptomyces HAases. Among these inhibitors, polymers of poly (styrene-4-sulfonate) (PSS) (i.e., molecular weight 1400-990,000 or PSS 1400-PSS 990,000) and O-sulfated HA (sHA) derivatives (sHA2.0, 2.5, and 2.75) were the most effective. HYAL-1 and bee HAases were the most sensitive, followed by testicular HAase; Streptomyces HAase was resistant to all inhibitors, except PSS 990,000 and VERSA-TL 502 (i.e., PSS 10(6) dalton). The length of the PSS polymer determined their potency (e.g., IC50 for HYAL-1, PSS 990,000: 0.0096 microM; PSS 210 no inhibition; IC50 for testicular HAase, PSS 990,000: 0.042 microM; PSS 210 no inhibition). The presence, but not the number, of sulfate groups on the sHA molecule determined its potency (e.g., IC50 for HYAL-1: sHA2.0, 0.019 microM; sHA2.75, 0.0083 microM). Other known HAase inhibitors, such as gossypol, sodium-aurothiomalate, 1-tetradecane sulfonic acid, and glycerrhizic acid, were not effective. Both PSS and sHA inhibited HAases by a mixed inhibition mechanism (i.e., competitive + uncompetitive) and were 5- to 17-fold better as uncompetitive inhibitors than as competitive inhibitors. These results demonstrate that HAase inhibitors show selectivity toward the different types of HAases, which could be exploited to inhibit specific HAases involved in a variety of pathophysiologic conditions.     

Network-based assessment of the selectivity of metabolic drug targets in Plasmodium falciparum with respect to human liver metabolism

ABSTRACT: BACKGROUND: The search for new drug targets for antibiotics against Plasmodium falciparum, a major cause of human deaths, is a pressing scientific issue, as multiple resistance strains spread rapidly. Metabolic network-based analyses may help to identify those parasite's essential enzymes whose homologous counterparts in the human host cells are either absent, non-essential or relatively less essential. RESULTS: Using the well-curated metabolic networks PlasmoNet of the parasite Plasmodium falciparum and HepatoNet1 of the human hepatocyte, the selectivity of 48 experimental antimalarial drug targets was analyzed. Applying in silico gene deletions, 24 of these drug targets were found to be perfectly selective, in that they were essential for the parasite but non-essential for the human cell. The selectivity of a subset of enzymes, that were essential in both models, was evaluated with the reduced fitness concept. It was, then, possible to quantify the reduction in functional fitness of the two networks under the progressive inhibition of the same enzymatic activity. Overall, this in silico analysis provided a selectivity ranking that was in line with numerous in vivo and in vitro observations. CONCLUSIONS: Genome-scale models can be useful to depict and quantify the effects of enzymatic inhibitions on the impaired production of biomass components. From the perspective of a host-pathogen metabolic interaction, an estimation of the drug targets-induced consequences can be beneficial for the development of a selective anti-parasitic drug.     

Factor VIIa inhibitors: gaining selectivity within the trypsin family

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.     

The selectivity of statine-based inhibitors against various human aspartic proteinases.

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1' positions. The residues occupying the peripheral sub-sites (P4 to P3') were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.     

The selectivity of inhibitors of protein kinase CK2: an update

CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.     

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors

Structure–activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC₅₀ 0.7 nM, FMS cell IC₅₀ 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.     

Development of a cheminformatics platform for selectivity analyses of carbonic anhydrase inhibitors

Abstract

The selectivity for a specific human Carbonic Anhydrase (hCA) isoform is an important property a hCA inhibitor (CAI) should be endowed with, in order to constitute a valuable therapeutic tool for the treatment of a desired pathology. In this context, we developed a chemoinformatic platform that allows the analysis of the structure and selectivity profile of known CAIs reported in literature, with the aim of identifying structural motifs connected to ligand selectivity, thus providing useful guidelines for the design of novel ligands selective for the desired hCA isoform. The platform is able to perform ultrafast structure and selectivity analyses through ligand fingerprint similarity, with no need of structural information about the target receptor and ligands’ binding mode. It is easily accessible to the non-expert user through the implementation of a KNIME Analytic Platform workflow and could be extended to analyze the selectivity profile of known ligands of different target proteins.     

Chiral linkers to improve selectivity of double-headed neuronal nitric oxide synthase inhibitors

To develop potent and selective nNOS inhibitors, new double-headed molecules with chiral linkers that derive from natural amino acids or their derivatives have been designed. The new structures contain two ether bonds, which greatly simplifies the synthesis and accelerates structure optimization. Inhibitor (R)-6b exhibits a potency of 32 nM against nNOS and is 475 and 244 more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 6b and the two heme propionates in nNOS, but not eNOS, is the structural basis for its high selectivity. This work demonstrates the importance of stereochemistry in this class of molecules, which significantly influences the potency and selectivity of the inhibitors. The structure–activity information gathered here provides a guide for future structure optimization.     

Implications for selectivity of 3,4-diarylquinolinones as p38alphaMAP kinase inhibitors

In this study we report on the specificity profiling of the MAP kinase inhibitors 1, 2, and 3 in a panel of 78 protein kinases including the MAPK isoforms p38(alpha,beta,gamma,delta), JNK1/2/3, and ERK1/2/8 showing 3-(4-fluorophenyl)-4-pyridin-4-ylquinolin-2(1H)-one (1) to be highly selective for p38alphaMAPK with an IC(50) of 1.8 microM. In contrast, besides p38alpha the isoxazoles 2 and 3 significantly inhibited JNK2/3 and further kinases beyond the MAPK family such as PKA, PKD, Lck, and CK1. By using sequence alignment and homology models of different members of the MAPK family the binding mode determining selectivity of 1 for the p38alpha isoform was investigated. For lead optimization of 1 a straightforward tandem-Buchwald-aldol synthetic approach toward the flexible decoration of the quinolin-2(1H)-one scaffold was employed. SAR for derivatives of 1 at the isolated p38alphaMAPK are presented.