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The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

Chakraborty S, Rendón-Ramírez A, Ásgeirsson B, Dutta M, Ghosh AS, Oda M, Venkatramani R, Rao BJ, Dandekar AM, Goñi FM - F1000Res (2013)

Bottom Line: The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies.However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs.Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400 005, India ; Plant Sciences Department, University of California, Davis, CA, 95616, USA.

ABSTRACT
The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.

No MeSH data available.


Related in: MedlinePlus

PI-PLC inhibition using DPP4 inhibitors.(a,c) Time courses of enzyme activity in the presence of varying amounts of inhibitors, respectively LAF-237 and K579. The trace marked LIPOSOMES corresponds to a control in the absence of PI-PLC. (b,d) Dose-response effect of inhibitors on PI-PLC activity. Activity was computed as the extent of vesicle aggregation after 10 min enzyme activity.
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f2: PI-PLC inhibition using DPP4 inhibitors.(a,c) Time courses of enzyme activity in the presence of varying amounts of inhibitors, respectively LAF-237 and K579. The trace marked LIPOSOMES corresponds to a control in the absence of PI-PLC. (b,d) Dose-response effect of inhibitors on PI-PLC activity. Activity was computed as the extent of vesicle aggregation after 10 min enzyme activity.

Mentions: Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) using dipeptidyl peptidase-IV (DPP4) inhibitors. DPP4 (EC 3.4.14.5), a serine protease that is expressed in many tissues (kidney, liver, lung, intestinal membranes, lymphocytes and endothelial cells), cleaves peptides with Pro or Ala residues in the second amino terminal position. Previously, we have experimentally demonstrated the existence of the serine protease domain in PI-PLC fromBacillus cereus - both by virtue of its proteolytic activity, and the inhibition of its native activity on phospholipids in the presence of serine protease inhibitors22. Furthermore, the specificity of the proteolytic activity indicated that it was a prolyl peptidase - thus, leading us to believe that DPP4 inhibitors should have a similar inhibitory effect on the PI-PLC enzyme.Table 1 shows the presence of a congruent motif in the PI-PLC protein with both Motif1 and Motif2. His32 and Asp67 are known to be a part of the active site scaffold in PI-PLC22. These proteins have completely different folds, and thus a superimposition (using both MUSTANG34 and DECAAF35) does not show any detectable similarity in their structures (Supplementary Figure 1).Figure 1 shows the active sites of these proteins, and the superimposition of these proteins based on their catalytic residues35. It can be seen that the closest non-polar residue to the catalytic triad in trypsin and PI-PLC (Ala56 in PDBid:1A0J, Ile68 in PDBid:1PTD) is differently placed from Val711 in DPP4 (PDBid:1N1M). This is also indicated by the greater RMSD (root mean square deviation) of the scaffold in PI-PLC to Motif2 as compared to Motif1. The differences in the position of peripheral residues is the source of the diverse specificities exhibited by these proteases.Figure 2 shows the inhibition of PI-PLC using two gliptins - vildagliptin (LAF-237)23 and K57924. PI-PLC catalyzes hydrolysis of phospholipids to yield diacylglycerol and a phosphoryl alcohol. In the absence of inhibitors enzyme addition to the vesicle suspension causes an increase in turbidity due to vesicle aggregation (Figure 2 a,c). Aggregation in turn occurs as a result of formation of the enzyme endproduct diacylglycerol36,37. A steady-state is reached under our conditions after 6–8 min. Addition of either LAF-237 (vildagliptin) or K579 leads to an obvious inhibition of the enzyme activity. Dose-response curves for the inhibitors are shown inFigure 2 (b,d). K579 is two orders of magnitude more potent than LAF-237 as a PI-PLC inhibitor, with half-maximal inhibitory concentrations IC50 respectively of 1µM and 100µM.


The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

Chakraborty S, Rendón-Ramírez A, Ásgeirsson B, Dutta M, Ghosh AS, Oda M, Venkatramani R, Rao BJ, Dandekar AM, Goñi FM - F1000Res (2013)

PI-PLC inhibition using DPP4 inhibitors.(a,c) Time courses of enzyme activity in the presence of varying amounts of inhibitors, respectively LAF-237 and K579. The trace marked LIPOSOMES corresponds to a control in the absence of PI-PLC. (b,d) Dose-response effect of inhibitors on PI-PLC activity. Activity was computed as the extent of vesicle aggregation after 10 min enzyme activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4309170&req=5

f2: PI-PLC inhibition using DPP4 inhibitors.(a,c) Time courses of enzyme activity in the presence of varying amounts of inhibitors, respectively LAF-237 and K579. The trace marked LIPOSOMES corresponds to a control in the absence of PI-PLC. (b,d) Dose-response effect of inhibitors on PI-PLC activity. Activity was computed as the extent of vesicle aggregation after 10 min enzyme activity.
Mentions: Inhibition of phosphoinositide-specific phospholipase C (PI-PLC) using dipeptidyl peptidase-IV (DPP4) inhibitors. DPP4 (EC 3.4.14.5), a serine protease that is expressed in many tissues (kidney, liver, lung, intestinal membranes, lymphocytes and endothelial cells), cleaves peptides with Pro or Ala residues in the second amino terminal position. Previously, we have experimentally demonstrated the existence of the serine protease domain in PI-PLC fromBacillus cereus - both by virtue of its proteolytic activity, and the inhibition of its native activity on phospholipids in the presence of serine protease inhibitors22. Furthermore, the specificity of the proteolytic activity indicated that it was a prolyl peptidase - thus, leading us to believe that DPP4 inhibitors should have a similar inhibitory effect on the PI-PLC enzyme.Table 1 shows the presence of a congruent motif in the PI-PLC protein with both Motif1 and Motif2. His32 and Asp67 are known to be a part of the active site scaffold in PI-PLC22. These proteins have completely different folds, and thus a superimposition (using both MUSTANG34 and DECAAF35) does not show any detectable similarity in their structures (Supplementary Figure 1).Figure 1 shows the active sites of these proteins, and the superimposition of these proteins based on their catalytic residues35. It can be seen that the closest non-polar residue to the catalytic triad in trypsin and PI-PLC (Ala56 in PDBid:1A0J, Ile68 in PDBid:1PTD) is differently placed from Val711 in DPP4 (PDBid:1N1M). This is also indicated by the greater RMSD (root mean square deviation) of the scaffold in PI-PLC to Motif2 as compared to Motif1. The differences in the position of peripheral residues is the source of the diverse specificities exhibited by these proteases.Figure 2 shows the inhibition of PI-PLC using two gliptins - vildagliptin (LAF-237)23 and K57924. PI-PLC catalyzes hydrolysis of phospholipids to yield diacylglycerol and a phosphoryl alcohol. In the absence of inhibitors enzyme addition to the vesicle suspension causes an increase in turbidity due to vesicle aggregation (Figure 2 a,c). Aggregation in turn occurs as a result of formation of the enzyme endproduct diacylglycerol36,37. A steady-state is reached under our conditions after 6–8 min. Addition of either LAF-237 (vildagliptin) or K579 leads to an obvious inhibition of the enzyme activity. Dose-response curves for the inhibitors are shown inFigure 2 (b,d). K579 is two orders of magnitude more potent than LAF-237 as a PI-PLC inhibitor, with half-maximal inhibitory concentrations IC50 respectively of 1µM and 100µM.

Bottom Line: The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies.However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs.Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400 005, India ; Plant Sciences Department, University of California, Davis, CA, 95616, USA.

ABSTRACT
The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.

No MeSH data available.


Related in: MedlinePlus