After 15-min incubation on ice, the films were taken out and 15?l of 60?mM NNDPD (3.6?mM last focus) and 15?l of 90?mM FeCl3 (5.4?mM last focus) were dispensed to each well (last quantity: 250?l). had been purified and portrayed from activity assays, isothermal calorimetry and X-ray crystallography, ensue to validate business lead give food to and substances details in to the pipeline for substance marketing. High-throughput H2S-detecting strategies have so far typically utilized the fluorescence assay predicated on the 7-azido-4-methylcoumarin (AzMC) dye. AzMC is reduced by H2S to 7-amino-4-methylcoumarin with concomitant upsurge in fluorescence33 selectively. Despite its specificity, AzMC not merely gradually reacts with H2S, but is also prone to chemical interference29. Other approaches have been recently reported based on non-commercially available H2S probes (e.g.34). The colorimetric methylene blue method is Fanapanel hydrate one of the most commonly used methods for H2S measurement. The method is based on the reaction of H2S with N,N-dimethyl-p-phenylenediamine (NNDPD), followed by iron chloride (FeCl3)-mediated formation of the methylene blue dye, which is usually detected by visible absorption spectroscopy35. Herein, we employed a combination of orthogonal biophysical and functional assays to screen a library of synthetic pyridine derivatives against the human H2S-synthesizing enzymes CBS, CSE and MST. Both SPR and DSF didnt detect any strongly interacting compound. An activity-based screening using the H2S-detecting AzMC fluorescent probe indicated positive hits. However, a counter-screen with the colorimetric methylene blue method revealed direct interference of the tested compounds with AzMC, thus rebutting the obtaining of positive hits, and allowed the identification of two compounds weakly inhibiting CBS and CSE. The experimental setup herein presented offers a robust platform for future compound screenings targeting the three human H2S-synthesizing enzymes. Results Synthesis of pyridine derivatives Thirty-one pyridine derivatives (Table?1 and Fig.?1) were synthesized and characterized as reported in the Materials and Methods section, employing condensation and heterocyclization reactions. Some of the prepared compounds were utilized for further transformation to water soluble salts. Compounds purity was at least 95%. Table 1 Pyridine derivatives screened against human H2S-synthesizing enzymes.
C1COOC2H5COONaCOOC2H5n/an/a 53 C2COOCH(CH3)2COONaCOOCH(CH3)2n/an/a 53 C3CNCOONaCNn/an/a 53 C4COCH3COONaCOCH3n/an/a 53 C5COOC2H5COOC2H5COOC2H5n/an/a 53 C6COOCH2COONaHCOOCH2COONan/an/a 54 C7COOCH2COOC2H5HCOOCH2COOC2H5n/an/a 54 C8COOCH2COONaCH3COOCH2COONan/an/a 54 C9COOCH2COOC2H5CH3COOCH2COOC2H5n/an/a 54 C10COOCH2COOC2H5C2H5COOCH2COOC2H5n/an/a 54 C11COOCH2COONaC2H5COOCH2COONan/an/a 54 C12COOC2H5CONHCH(COONa)(CH2)2COONaCOOC2H5n/an/a 51 C13COOC2H5CONH(CH2)2SO3NaCOOC2H5n/an/a 51 C14COOC2H5CONH(CH2)3COONaCOOC2H5n/an/a 51 C15COOC2H5CONH(CH2)2COOHCOOC2H5n/an/a 51 C16COOC2H5COOCH2CONH2COOC2H5n/an/aThis workC17COOC2H5COOCH2COOC2H5COOC2H5n/an/aThis workC18COOC2H5COOCH2COC6H5COOC2H5n/an/aThis workC19COOC2H5COOCH2COC6H4OCH3-4COOC2H5n/an/aThis workC20n/an/an/an/an/a 56 C21COO(CH2)2COONaCOOCH3COO(CH2)2COONan/an/a 55 C22COOCH2COOCH3COOHCOOCH2COOCH3n/an/a 55 C23COOCH2COOC2H5thienylCOOCH2COOC2H5n/an/a 55 C24COOCH2COONathienylCOOCH2COONan/an/a 55 C25n/an/an/aCOOHC2H5This workC26n/an/an/aCOOC2H5C2H5This workC27n/an/an/athienylCH2COOC2H5This workC28n/an/an/athienylCH2COONaThis workC29n/an/an/an/an/a 52 C30n/an/an/an/an/a 50 C31n/an/an/an/an/a 50 Open in a separate window Open in a separate window Physique 1 Chemical structures of pyridine derivatives. Conversation with human CBS, CSE and MST analyzed by differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR) The newly synthesized derivatives were assayed by two complementary biophysical techniques, namely DSF and SPR, for their ability to bind to the H2S-synthesizing human enzymes tCBS, CSE and MST, recombinantly expressed and purified from E. coli. For each target protein, the DSF assays were preliminarily optimized in terms of protein and dye concentration, resulting in the following conditions (final volume: 20?L in each well): tCBS (2?g/well; ~2?M), CSE (1?g/well; ~1?M) or MST (2?g/well; ~3?M); final dye concentration: 1x. As shown in Fig.?2 (top panel), the DSF thermal denaturation curve of tCBS (marked with a) displays an unusually high fluorescence of the dye at the initial heat (20?C), indicating either a partial unfolding of the protein or a possible interference from a protein component in the assay. Data were best fitted (collection a in Fig.?2, top panel) with two consecutive transitions with very close values: Tm1?=?45.7?C (60%) and Tm2?=?49.8?C (40%), yielding a weighted mean value Tm_Ave?=?47.4?C. To check whether tCBS was partly unfolded at the initial heat of 20?C, thermal unfolding was also monitored by Far-UV CD spectropolarimetry. As shown in Fig.?2 (top panel), according to the CD thermal denaturation profile acquired at 222?nm (marked with b), there is no indication of denatured protein at the initial heat of 20?C. Moreover, the protein shows an obvious Tm of 58.6?C. DSF was used to screen the result from the pyridine derivatives at 200?M focus on the thermal denaturation profile of tCBS. Statistical validation from the assay was obtained by incubating with 200 tCBS?M AOAA mainly because the adverse control (N?=?25; 6 3rd party experiments), to become weighed against the non-incubated enzyme as positive control (N?=?23; 6 3rd party tests) (Supplementary Fig.?Supplementary and S1 Table?S1). The assay shown Z-factors of ?1.58,?+?0.08 and ?0.28 for Tm1, Tm_Ave and Tm2, respectively. Substances C9, C10, C19 and C23 led to aberrant thermal denaturation information, precluding any kind of analysis. The rest of the substances had a restricted effect on the tCBS thermal denaturation (Supplementary Fig.?S2). While non-e from the substances exhibited |Z-score|??3.0 for just about any from the analysed guidelines (Tm1, Tm2 and Tm_Ave),.The microplate was incubated at 4?C for 2?hours to favour possible relationships with the prospective proteins. concomitant upsurge in fluorescence33. Despite its specificity, AzMC not merely reacts with H2S gradually, but can be prone to chemical substance interference29. Other techniques have been lately reported predicated on non-commercially obtainable H2S probes (e.g.34). The colorimetric methylene blue technique is among the most commonly utilized options for H2S dimension. The method is dependant on the result of H2S with N,N-dimethyl-p-phenylenediamine (NNDPD), accompanied by iron chloride (FeCl3)-mediated development from the methylene blue dye, which can be detected by noticeable absorption spectroscopy35. Herein, we used a combined mix of orthogonal biophysical and practical assays to display a collection of artificial pyridine derivatives against the human being H2S-synthesizing enzymes CBS, CSE and MST. Both SPR and DSF didnt identify any highly interacting substance. An activity-based testing using the H2S-detecting AzMC fluorescent probe indicated positive strikes. Nevertheless, a counter-screen using the colorimetric methylene blue technique revealed direct disturbance from the examined substances with AzMC, therefore rebutting the locating of positive strikes, and allowed the recognition of two substances weakly inhibiting CBS and CSE. The experimental set up herein presented gives a robust system for future substance screenings focusing on the three human being H2S-synthesizing enzymes. Outcomes Synthesis of pyridine derivatives Thirty-one pyridine derivatives (Desk?1 and Fig.?1) were synthesized and characterized while reported in the Components and Strategies section, employing condensation and heterocyclization reactions. A number of the ready substances were useful for additional transformation to drinking water soluble salts. Substances purity was at least 95%. Desk 1 Pyridine derivatives screened against human being H2S-synthesizing enzymes.
C1COOC2H5COONaCOOC2H5n/an/a 53 C2COOCH(CH3)2COONaCOOCH(CH3)2n/an/a 53 C3CNCOONaCNn/an/a 53 C4COCH3COONaCOCH3n/an/a 53 C5COOC2H5COOC2H5COOC2H5n/an/a 53 C6COOCH2COONaHCOOCH2COONan/an/a 54 C7COOCH2COOC2H5HCOOCH2COOC2H5n/an/a 54 C8COOCH2COONaCH3COOCH2COONan/an/a 54 C9COOCH2COOC2H5CH3COOCH2COOC2H5n/an/a 54 C10COOCH2COOC2H5C2H5COOCH2COOC2H5n/an/a 54 C11COOCH2COONaC2H5COOCH2COONan/an/a 54 C12COOC2H5CONHCH(COONa)(CH2)2COONaCOOC2H5n/an/a 51 C13COOC2H5CONH(CH2)2SO3NaCOOC2H5n/an/a 51 C14COOC2H5CONH(CH2)3COONaCOOC2H5n/an/a 51 C15COOC2H5CONH(CH2)2COOHCOOC2H5n/an/a 51 C16COOC2H5COOCH2CONH2COOC2H5n/an/aThis workC17COOC2H5COOCH2COOC2H5COOC2H5n/an/aThis workC18COOC2H5COOCH2COC6H5COOC2H5n/an/aThis workC19COOC2H5COOCH2COC6H4OCH3-4COOC2H5n/an/aThis workC20n/an/an/an/an/a 56 C21COO(CH2)2COONaCOOCH3COO(CH2)2COONan/an/a 55 C22COOCH2COOCH3COOHCOOCH2COOCH3n/an/a 55 C23COOCH2COOC2H5thienylCOOCH2COOC2H5n/an/a 55 C24COOCH2COONathienylCOOCH2COONan/an/a 55 C25n/an/an/aCOOHC2H5This workC26n/an/an/aCOOC2H5C2H5This workC27n/an/an/athienylCH2COOC2H5This workC28n/an/an/athienylCH2COONaThis workC29n/an/an/an/an/a 52 C30n/an/an/an/an/a 50 C31n/an/an/an/an/a 50 Open up in another window Open up in another window Shape 1 Chemical constructions of pyridine derivatives. Discussion with human being CBS, CSE and MST examined by differential checking fluorimetry (DSF) and surface area plasmon resonance (SPR) The recently synthesized derivatives had been assayed by two complementary biophysical methods, specifically DSF and SPR, for his or her capability to bind towards the H2S-synthesizing human being enzymes tCBS, CSE and MST, recombinantly indicated and purified from E. coli. For every target proteins, the DSF assays had been preliminarily optimized with regards to proteins and dye focus, leading to the following circumstances (final quantity: 20?L in each well): tCBS (2?g/well; ~2?M), CSE (1?g/well; ~1?M) or MST (2?g/well; ~3?M); last dye focus: 1x. As demonstrated in Fig.?2 (best -panel), the DSF thermal denaturation curve of tCBS (marked having a) shows an unusually high fluorescence from the dye in the initial temperatures (20?C), indicating the partial unfolding from the proteins or a feasible disturbance from a proteins element in the assay. Data had been best installed (range a in Fig.?2, best -panel) with two consecutive transitions with very close ideals: Tm1?=?45.7?C (60%) and Tm2?=?49.8?C (40%), yielding a weighted mean worth Tm_Ave?=?47.4?C. To check on whether tCBS was partially unfolded at the original temperatures of 20?C, thermal unfolding was also monitored by Far-UV Compact disc spectropolarimetry. As demonstrated in Fig.?2 (best panel), based on the Compact disc thermal denaturation profile acquired in 222?nm (marked with b), there is absolutely no indicator of denatured proteins in the initial temperatures of 20?C. Furthermore, the proteins displays an apparent Tm of 58.6?C. DSF was used to screen the effect of the pyridine derivatives at 200?M concentration on the thermal denaturation profile of tCBS. Statistical validation of the assay was acquired by incubating tCBS with 200?M AOAA mainly because the bad control (N?=?25; 6 self-employed experiments), to be compared with the non-incubated enzyme as positive control (N?=?23; 6 self-employed experiments) (Supplementary Fig.?S1 and Supplementary Table?S1). The assay displayed Z-factors of ?1.58,?+?0.08 and ?0.28 for Tm1, Tm2 and Tm_Ave, respectively. Compounds C9, C10, C19 and C23 resulted in aberrant thermal denaturation profiles, precluding any type of analysis. The remaining compounds had a limited impact on the tCBS thermal denaturation (Supplementary Fig.?S2). While none of the compounds exhibited |Z-score|??3.0 for any of the analysed guidelines (Tm1, Tm2 and Tm_Ave), four compounds, namely C2, C14, C28 and C29, yielded |Tm_Ave|??1.0?C. Open in a separate window Number 2 Thermal denaturation profiles of human being H2S-synthesizing enzymes. Thermal denaturation profiles of recombinant human being cystathionine.Modified H2S metabolism is definitely associated with multiple human being pathologies, such as cardiovascular17 and inflammatory42 disorders, neurodegeneration19 and cancer22,43. probes (e.g.34). The colorimetric methylene blue method is one of the most commonly used methods for H2S measurement. The method is based on the reaction of H2S with N,N-dimethyl-p-phenylenediamine (NNDPD), followed by iron chloride (FeCl3)-mediated formation of the methylene blue dye, which is definitely detected by visible absorption spectroscopy35. Herein, we used a combination of orthogonal biophysical and practical assays to display a library of synthetic pyridine derivatives against the human being H2S-synthesizing enzymes CBS, CSE and MST. Both SPR and DSF didnt detect any strongly interacting compound. An activity-based screening using the H2S-detecting AzMC fluorescent probe indicated positive hits. However, a counter-screen with the colorimetric methylene blue method revealed direct interference of the tested compounds with AzMC, therefore rebutting the getting of positive hits, and allowed the recognition of two compounds weakly inhibiting CBS and CSE. The experimental setup herein presented gives a robust platform for future compound screenings focusing on the three human being H2S-synthesizing enzymes. Results Synthesis of pyridine derivatives Thirty-one pyridine derivatives (Table?1 and Fig.?1) were synthesized and characterized while reported in the Materials and Methods section, employing condensation and heterocyclization reactions. Some of the prepared compounds were utilized for further transformation to water soluble salts. Compounds purity was at least 95%. Table 1 Pyridine derivatives screened against human being H2S-synthesizing enzymes.
C1COOC2H5COONaCOOC2H5n/an/a 53 C2COOCH(CH3)2COONaCOOCH(CH3)2n/an/a 53 C3CNCOONaCNn/an/a 53 C4COCH3COONaCOCH3n/an/a 53 C5COOC2H5COOC2H5COOC2H5n/an/a 53 C6COOCH2COONaHCOOCH2COONan/an/a 54 C7COOCH2COOC2H5HCOOCH2COOC2H5n/an/a 54 C8COOCH2COONaCH3COOCH2COONan/an/a 54 C9COOCH2COOC2H5CH3COOCH2COOC2H5n/an/a 54 C10COOCH2COOC2H5C2H5COOCH2COOC2H5n/an/a 54 C11COOCH2COONaC2H5COOCH2COONan/an/a 54 C12COOC2H5CONHCH(COONa)(CH2)2COONaCOOC2H5n/an/a 51 C13COOC2H5CONH(CH2)2SO3NaCOOC2H5n/an/a 51 C14COOC2H5CONH(CH2)3COONaCOOC2H5n/an/a 51 C15COOC2H5CONH(CH2)2COOHCOOC2H5n/an/a 51 C16COOC2H5COOCH2CONH2COOC2H5n/an/aThis workC17COOC2H5COOCH2COOC2H5COOC2H5n/an/aThis workC18COOC2H5COOCH2COC6H5COOC2H5n/an/aThis workC19COOC2H5COOCH2COC6H4OCH3-4COOC2H5n/an/aThis workC20n/an/an/an/an/a 56 C21COO(CH2)2COONaCOOCH3COO(CH2)2COONan/an/a 55 C22COOCH2COOCH3COOHCOOCH2COOCH3n/an/a 55 C23COOCH2COOC2H5thienylCOOCH2COOC2H5n/an/a 55 C24COOCH2COONathienylCOOCH2COONan/an/a 55 C25n/an/an/aCOOHC2H5This workC26n/an/an/aCOOC2H5C2H5This workC27n/an/an/athienylCH2COOC2H5This workC28n/an/an/athienylCH2COONaThis workC29n/an/an/an/an/a 52 C30n/an/an/an/an/a 50 C31n/an/an/an/an/a 50 Open in a separate window Open in a separate window Number 1 Chemical constructions of pyridine derivatives. Connection with human being CBS, CSE and MST analyzed by differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR) The newly synthesized derivatives were assayed by two complementary biophysical techniques, namely DSF and SPR, for his or her ability to bind to the H2S-synthesizing human being enzymes tCBS, CSE and MST, recombinantly indicated and purified from E. coli. For each target protein, the DSF assays were preliminarily optimized in terms of protein and dye concentration, resulting in the following conditions (final volume: Fanapanel hydrate 20?L in each well): tCBS (2?g/well; ~2?M), CSE (1?g/well; ~1?M) or MST (2?g/well; ~3?M); final dye concentration: 1x. As demonstrated in Fig.?2 (top panel), the DSF thermal denaturation curve of tCBS (marked using a) shows an unusually high fluorescence from the dye in the initial heat range (20?C), indicating the partial unfolding from the proteins or a feasible disturbance from a proteins element in the assay. Data had been best installed (series a in Fig.?2, best -panel) with two consecutive transitions with very close beliefs: Tm1?=?45.7?C (60%) and Tm2?=?49.8?C (40%), yielding a weighted mean worth Tm_Ave?=?47.4?C. To check on whether tCBS was partially unfolded at the original heat range of 20?C, thermal unfolding was also monitored by Far-UV Compact disc spectropolarimetry. As proven in Fig.?2 (best panel), based on the Compact disc thermal denaturation profile acquired in 222?nm (marked with b), there is absolutely no sign of denatured proteins in the initial heat range of 20?C. Furthermore, the proteins shows an obvious Tm of 58.6?C. DSF was utilized to screen the result from the pyridine derivatives at 200?M focus on the thermal denaturation profile of tCBS. Statistical validation from the assay was attained by incubating tCBS with 200?M AOAA simply because.H2O: C, 59.07; H, 7.13; N, 4.30. Despite its specificity, AzMC not merely reacts with H2S gradually, but can be prone to chemical substance interference29. Other strategies have been lately reported predicated on non-commercially obtainable H2S probes (e.g.34). The colorimetric methylene blue technique is among the most commonly utilized options for H2S dimension. The method is dependant on the result of H2S with N,N-dimethyl-p-phenylenediamine (NNDPD), accompanied by iron chloride (FeCl3)-mediated development from the methylene blue dye, which is normally detected by noticeable absorption spectroscopy35. Herein, we utilized a combined mix of orthogonal biophysical and useful assays to display screen a collection of artificial pyridine derivatives against the individual H2S-synthesizing enzymes CBS, CSE and MST. Both SPR and DSF didnt identify any highly interacting substance. An activity-based testing using the H2S-detecting AzMC fluorescent probe indicated positive strikes. Nevertheless, a counter-screen using the colorimetric methylene blue technique revealed direct disturbance from the examined substances with AzMC, hence rebutting the selecting of positive strikes, and allowed the id of two substances weakly inhibiting CBS and CSE. The experimental set up herein presented presents a robust system for future substance screenings concentrating on the three individual H2S-synthesizing enzymes. Outcomes Synthesis of pyridine derivatives Thirty-one pyridine derivatives (Desk?1 and Fig.?1) were synthesized and characterized seeing that reported in the Components and Strategies section, employing condensation and heterocyclization reactions. A number of the ready substances were employed for additional transformation to drinking water soluble salts. Substances purity was at least 95%. Desk 1 Pyridine derivatives screened against individual H2S-synthesizing enzymes.
C1COOC2H5COONaCOOC2H5n/an/a 53 C2COOCH(CH3)2COONaCOOCH(CH3)2n/an/a 53 C3CNCOONaCNn/an/a 53 C4COCH3COONaCOCH3n/an/a 53 C5COOC2H5COOC2H5COOC2H5n/an/a 53 C6COOCH2COONaHCOOCH2COONan/an/a 54 C7COOCH2COOC2H5HCOOCH2COOC2H5n/an/a 54 C8COOCH2COONaCH3COOCH2COONan/an/a 54 C9COOCH2COOC2H5CH3COOCH2COOC2H5n/an/a 54 C10COOCH2COOC2H5C2H5COOCH2COOC2H5n/an/a 54 C11COOCH2COONaC2H5COOCH2COONan/an/a 54 C12COOC2H5CONHCH(COONa)(CH2)2COONaCOOC2H5n/an/a 51 C13COOC2H5CONH(CH2)2SO3NaCOOC2H5n/an/a 51 C14COOC2H5CONH(CH2)3COONaCOOC2H5n/an/a 51 C15COOC2H5CONH(CH2)2COOHCOOC2H5n/an/a 51 C16COOC2H5COOCH2CONH2COOC2H5n/an/aThis workC17COOC2H5COOCH2COOC2H5COOC2H5n/an/aThis workC18COOC2H5COOCH2COC6H5COOC2H5n/an/aThis workC19COOC2H5COOCH2COC6H4OCH3-4COOC2H5n/an/aThis workC20n/an/an/an/an/a 56 C21COO(CH2)2COONaCOOCH3COO(CH2)2COONan/an/a 55 C22COOCH2COOCH3COOHCOOCH2COOCH3n/an/a 55 C23COOCH2COOC2H5thienylCOOCH2COOC2H5n/an/a 55 C24COOCH2COONathienylCOOCH2COONan/an/a 55 C25n/an/an/aCOOHC2H5This workC26n/an/an/aCOOC2H5C2H5This workC27n/an/an/athienylCH2COOC2H5This workC28n/an/an/athienylCH2COONaThis workC29n/an/an/an/an/a 52 C30n/an/an/an/an/a 50 C31n/an/an/an/an/a 50 Open up in another window Open up in another window Amount 1 Chemical buildings of pyridine derivatives. Connections with individual CBS, CSE and MST examined by differential checking fluorimetry (DSF) and surface area plasmon resonance (SPR) The recently synthesized derivatives had been assayed by two complementary biophysical methods, specifically DSF and SPR, because of their capability to bind towards the H2S-synthesizing individual enzymes tCBS, CSE and MST, recombinantly portrayed and purified from E. coli. For every target proteins, the DSF assays had been preliminarily optimized with regards to proteins and dye focus, leading to the following circumstances (final quantity: 20?L in each well): tCBS (2?g/well; ~2?M), CSE (1?g/well; ~1?M) or MST (2?g/well; ~3?M); last dye focus: 1x. As proven in Fig.?2 (best -panel), the DSF thermal Fanapanel hydrate denaturation curve of tCBS (marked using a) shows an unusually high fluorescence of the dye at the initial temperature (20?C), indicating either a partial unfolding of the protein or a possible interference from a protein component in the assay. Data were best fitted (line a in Fig.?2, top panel) with two consecutive transitions with very close values: Tm1?=?45.7?C (60%) and Tm2?=?49.8?C (40%), yielding a weighted mean value Tm_Ave?=?47.4?C. To check whether tCBS was partly unfolded at the initial temperature of 20?C, thermal unfolding was also monitored by Far-UV CD spectropolarimetry. As shown in Fig.?2 (top panel), according to the CD thermal denaturation profile acquired at 222?nm (marked with b), there is no indication of denatured protein at the initial temperature of 20?C. Moreover, the protein displays an apparent Tm of 58.6?C. DSF was employed to screen the effect of the pyridine derivatives at 200?M concentration on the thermal denaturation profile of tCBS. Statistical validation of the assay was obtained by incubating tCBS with 200?M AOAA as the unfavorable control.SPR sensorgrams obtained for the conversation between immobilized recombinant human cystathionine -synthase (CBS, top panel), cystathionine -lyase (CSE, middle panel) or 3-mercaptopyruvate sulfurtransferase (MST, bottom panel) with the indicated pyridine derivatives at 25, 50, 100 and 200?M. Inhibition of human CBS, CSE and MST analyzed by fluorimetric and colorimetric assays The screening was complemented with activity measurements to test the inhibitory efficacy of the synthetic compounds towards the target enzymes. concomitant increase in fluorescence33. Despite its specificity, AzMC not only reacts with H2S slowly, but is also prone to chemical interference29. Other approaches have been recently reported based on non-commercially available H2S probes (e.g.34). The colorimetric methylene blue method is one of the most commonly used methods for H2S measurement. The method is based on the reaction of H2S with N,N-dimethyl-p-phenylenediamine (NNDPD), followed by iron chloride (FeCl3)-mediated formation of the methylene blue dye, which is usually detected by visible absorption spectroscopy35. Herein, we employed a combination of orthogonal biophysical and functional assays to screen a library of synthetic pyridine derivatives against the human H2S-synthesizing enzymes CBS, CSE and MST. Both SPR and DSF didnt detect any strongly interacting compound. An activity-based screening using the H2S-detecting AzMC fluorescent probe indicated positive hits. However, a counter-screen with the colorimetric methylene blue method revealed direct interference of the tested compounds with AzMC, thus rebutting the obtaining of positive hits, and allowed the identification of two compounds weakly inhibiting CBS and CSE. The experimental setup herein presented offers a robust platform for future compound screenings targeting the three human H2S-synthesizing enzymes. Results Synthesis of pyridine derivatives Thirty-one pyridine derivatives (Table?1 and Fig.?1) were synthesized and characterized as reported in the Materials and Methods section, employing condensation and heterocyclization reactions. Some of the prepared compounds were used for further transformation to water soluble salts. Compounds purity was at least 95%. Table 1 Pyridine derivatives screened against human H2S-synthesizing enzymes.
C1COOC2H5COONaCOOC2H5n/an/a 53 C2COOCH(CH3)2COONaCOOCH(CH3)2n/an/a 53 C3CNCOONaCNn/an/a 53 C4COCH3COONaCOCH3n/an/a 53 C5COOC2H5COOC2H5COOC2H5n/an/a 53 C6COOCH2COONaHCOOCH2COONan/an/a 54 C7COOCH2COOC2H5HCOOCH2COOC2H5n/an/a 54 C8COOCH2COONaCH3COOCH2COONan/an/a 54 C9COOCH2COOC2H5CH3COOCH2COOC2H5n/an/a 54 C10COOCH2COOC2H5C2H5COOCH2COOC2H5n/an/a 54 C11COOCH2COONaC2H5COOCH2COONan/an/a 54 C12COOC2H5CONHCH(COONa)(CH2)2COONaCOOC2H5n/an/a 51 C13COOC2H5CONH(CH2)2SO3NaCOOC2H5n/an/a 51 C14COOC2H5CONH(CH2)3COONaCOOC2H5n/an/a 51 C15COOC2H5CONH(CH2)2COOHCOOC2H5n/an/a 51 C16COOC2H5COOCH2CONH2COOC2H5n/an/aThis workC17COOC2H5COOCH2COOC2H5COOC2H5n/an/aThis workC18COOC2H5COOCH2COC6H5COOC2H5n/an/aThis workC19COOC2H5COOCH2COC6H4OCH3-4COOC2H5n/an/aThis workC20n/an/an/an/an/a 56 C21COO(CH2)2COONaCOOCH3COO(CH2)2COONan/an/a 55 C22COOCH2COOCH3COOHCOOCH2COOCH3n/an/a 55 C23COOCH2COOC2H5thienylCOOCH2COOC2H5n/an/a 55 C24COOCH2COONathienylCOOCH2COONan/an/a 55 C25n/an/an/aCOOHC2H5This workC26n/an/an/aCOOC2H5C2H5This workC27n/an/an/athienylCH2COOC2H5This workC28n/an/an/athienylCH2COONaThis workC29n/an/an/an/an/a 52 C30n/an/an/an/an/a 50 C31n/an/an/an/an/a 50 Open in a separate window Open in a separate window Figure 1 Chemical structures of pyridine derivatives. Interaction with human CBS, CSE and MST analyzed by differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR) The newly synthesized derivatives were assayed by two complementary biophysical techniques, namely DSF and SPR, for their ability to bind to the H2S-synthesizing human enzymes tCBS, CSE and MST, recombinantly expressed and purified from E. coli. For each target protein, the DSF assays were preliminarily optimized in terms of protein and dye concentration, resulting in the following conditions (final volume: 20?L in each well): tCBS (2?g/well; ~2?M), CSE (1?g/well; ~1?M) or MST (2?g/well; ~3?M); final dye concentration: 1x. As shown in Fig.?2 (top panel), the DSF thermal denaturation curve of tCBS (marked with SIGLEC6 a) displays an unusually high fluorescence of the dye at the initial temperature (20?C), indicating either a Fanapanel hydrate partial unfolding of the protein or a possible interference from a protein component in the assay. Data were best fitted (line a in Fig.?2, top panel) with two consecutive transitions with very close values: Tm1?=?45.7?C (60%) and Tm2?=?49.8?C (40%), yielding a weighted mean value Tm_Ave?=?47.4?C. To check whether tCBS was partly unfolded at the initial temperature of 20?C, thermal unfolding was also monitored by Far-UV CD spectropolarimetry. As shown in Fig.?2 (top panel), according to the CD thermal denaturation profile acquired at 222?nm (marked with b), there is no indication of denatured protein at the initial temperature of 20?C. Moreover, the protein displays an apparent Tm of 58.6?C. DSF was employed.
