Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

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.

Oral glucose elicits a greater insulin response than intravenous glucose infusion, a phenomenon known as the incretin effect 1 . This effect is mostly attributed to the intestinally derived hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) 2 . These hormones have a very short half-life as they are rapidly inactivated by the ubiquitous enzyme dipeptidyl peptidase-IV (DPP4) 3 . The finding that the incretin effect is impaired in subjects with type 2 diabetes 4 led to two major types of GLP-1 based therapies 5 -intravenously or sub-cutaneously administered GLP-1 mimetics that are resistant to DPP4 (exenatide, liraglutide, etc.) 6 , and the orally administered gliptins that prolong the physiological actions of incretin hormones by inhibiting DPP4 (sitagliptin, vildagliptin, etc.) [7][8][9] . Due to the multifarious roles played by the DPP4 enzyme 10-12 , the possible side effects of these drugs (acute pancreatitis, pancreatic cancer, etc. [13][14][15] ) are strongly contested by researchers who argue that current statistics are insufficient 16,17 to conclusively attribute these side effects to the otherwise beneficial GLP-1 drugs 18 . Compound promiscuity is another phenomenon that might play a crucial role in determining the side effects of these therapies, although this aspect has rarely been pursued intensively 19 .
Previous work by our group has established the spatial and electrostatic congruence in cognate residue pairs of the active site in proteins with the same functionality (CLASP) 20,21 . CLASP analysis indicated that the phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus has spatial and electrostatic congruence with a serine protease motif 22 . This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC 50 = 0.018 mM). The specificity of the protease activity was for a proline in the amino terminal, suggesting that PI-PLC is a prolyl peptidase, similar to the DPP4 enzyme. This finding led us to believe that the gliptins would have similar inhibitory effect on PI-PLC. In the current work, we have confirmed the inhibition of the native phospholipase activity of PI-PLC using two gliptins -vildagliptin 23 (at µ-molar concentrations) and K579 24 (at nano-molar concentrations).
Subsequently, we used a motif derived from a DPP4 protein 25 , in addition to the trypsin motif used previously 22 , to query a comprehensive and non-redundant (50% sequence identity) list of ~5000 human proteins with known structures using CLASP, intending to identify other proteins that might be inhibited by the gliptins. From the set of proteins with significant congruent matches with these two motifs, we identified a pancreatic lipase 26 and a gastric lipase 27 , keeping the context of lipases, acute pancreatitis and GLP-1 based therapies in mind. Our findings rationalize the elevated levels of serum lipase found in patients undergoing DPP4 inhibitor based therapies 28,29 , although these reports are in disagreement with other findings 30,31 . While it is logical and expected to find scaffolds that are congruent to trypsin and DPP4 active sites in lipases based on the current results and our previous findings 22 , we also show the presence of the serine catalytic triad in close proximity to the active site residues of proteins which have a completely different enzymatic mechanism (for example, in glutaminyl cyclase which is a transferase 32 ). This corroborates the current belief that convergent evolution occurs more frequently than previously believed 33 . Thus, we propose a rational method to identify proteins that might have unintended and undesirable interactions with newly introduced compounds, and substantiate our claims by demonstrating the inhibition of the native phospholipase activity of PI-PLC from B. cereus using gliptins that are used in type 2 diabetes therapy.

Results
The active site motifs The active sites of serine proteases differ in their specificities owing to residues other than the conserved catalytic triad. Thus, in addition to the trypsin motif used previously (Asp102, Ser195 and His57 -PDBid 1A0J) 22 (Motif1), we choose another motif from a DPP4 enzyme (Asp708, Ser630 and His740 -PDBid:1N1M) (Motif2) ( Table 1). Apart from the catalytic triad, we chose another non-polar residue in order to increase the specificity of the matches (Ala56 in Motif1 and Val711 in Motif2). This fourth residue is chosen as the closest residue to any one of the catalytic triad residues. Using the ability of CLASP to include stereochemically equivalent residues, this last residue could be matched by another non-polar residue -one of Gly, Ala, Val, Leu, Ile or Met. Further, it has been seen that the second (ac) and fifth (bd) ( Table 1) pairwise electrostatic potential differences (EPD) are not discriminatory -thus, this pair is not used to score the EPD difference (although it is included in the distance deviation score). 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 from Bacillus cereus -both by virtue of its proteolytic activity, and the inhibition of its native activity on phospholipids in the presence of serine protease inhibitors 22 . 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-PLC 22 . These proteins have completely different folds, and thus a superimposition (using both MUSTANG 34 and DECAAF 35 ) 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

Amendments from Version 2
In the current version, we have changed the title, and cited previous research (ref 41 and 54) based on referee suggestions.
We have also included some minor corrections as suggested by a co-author. TRYPSIN (1A0J) D102,S195 H57,A56 based on their catalytic residues 35 . It can be seen that the closest non-polar residue to the catalytictriad 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 K579 24 . 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 diacylglycerol 36,37 . A steady-state is reached under our conditions after 6-8 min. Addition of either  or K579 leads to an obvious inhibition of the enzyme activity.

REVISED
Dose-response curves for the inhibitors are shown in Figure 2 (b,d).
K579 is two orders of magnitude more potent than LAF-237 as a PI-PLC inhibitor, with half-maximal inhibitory concentrations IC 50 respectively of 1 µM and 100 µM.
Phosphoinositide-specific phospholipase C inhibition data using the dipeptidyl peptidase-IV inhibitors K-579 and LAF-237    Table 1 shows the congruence of the DPP4 motif to these proteins using Motif1 and Motif2. It is interesting to note that the gastric lipase (PDBid:1HLG) has a good match with both motifs -Leu326 in PDBid:1HLG is congruent to Ala56 in PDBid:1A0J, and Ala237 (PDBid:1HLG) is congruent to Val711 (PDBid:1N1M).
Since both these proteins are lipases (hydrolases), this congruence to Motif1 and Motif2 is expected based on our previous results with PI-PLC 22 . However, our methodology also detects other proteins, often with a completely different enzymatic mechanism from hydrolases. A glutaminyl cyclase (PDBid:3PB4 32 ), a transferase, has a significantly congruent domain with Motif1 (lesser congruence with Motif2, as indicated by the RMSD) ( Table 1). Figure 3 shows the proximity of the promiscuous scaffold to the active site of the cyclase, and also the congruence of the scaffold to Motif1.    ligand-DPP4 structure is solved (Supplementary Table 2). For uniformity, we chose the first four closest atoms from the protein that make contacts to the ligand, excluding hydrophobic interactions. Table 3 shows the number of times each residue in DPP4 makes contact to the ligand. Three residues are ubiquitous in making contacts in all these ligands: Glu205, Glu206 and Tyr662 made contacts in 71, 68 and 63 ligands, respectively. Interestingly, Glu205 and Glu206 have been implicated as critical residues for the enzymatic activity of DPP4 through point mutations 41 . Note, that since only the first four residues were considered, these counts are conservative (and might be more). A recent study has found that inhibitors that bind to residues beyond the extensive subsite (defined as Val207, Ser209, Phe357 and Arg358) increases DPP4 inhibition, as compared to those inhibitors that form a covalent bond with Ser630 38 . Table 3 shows that very few inhibitors make such contacts. We created a library of motifs from these structures that can be used to query any protein using CLASP to determine the possibility that DPP4 inhibitors might bind to it (Supplementary Table 3), after removing equivalent ones to eliminate redundancy. This table shows the final list of 39 motifs (pruned from the initial 76): this is a comprehensive set of motifs that encapsulates the current knowledge about protein ligand interactions for the DPP4 enzyme. A facet of ligand binding that needs to be accounted for while choosing a motif is the spatial and electrostatic changes that can be induced by ligand binding. Thus, we obtain the residues involved in binding from the holo enzyme, but extract the motif values (pairwise distance and EPD) from the apo enzyme.

Discussion
The controversy regarding the side effects of the dpp4 inhibitors, particularly with respect to acute pancreatitis and pancreatic cancer, continues unabated. While some researchers feel that it is not acceptable to assume that 'absence of evidence is evidence of absence' 42,43 , others believe that current data are not conclusive and the 'benefits by far outweigh the potential risks' 16 . Adding to the uncertainties are conflicting reports presented by different groups [28][29][30][31] . Notwithstanding the antagonistic views on the subject, it is unanimously accepted that current data are insufficient to establish a causal pathogenic effect of these drugs on such side effects 44 . Various database studies have been undertaken in order to ascertain the effects of the GLP-1 therapies. Some studies 'did not find an association between the use of exenatide or sitagliptin and acute pancreatitis' with the caveat that the 'limitations of this observational claims-based analysis cannot exclude the possibility of an increased risk' 45 . On the other hand, other studies have shown that the use of 'sitagliptin or exenatide increased the odds ratio for reported pancreatitis 6-fold as compared with other therapies' 14 . Further, they reported that 'pancreatic cancer was more commonly reported among patients who took sitagliptin or exenatide as compared with other therapies' 14 . Although these studies concern the usage of both GLP-1 mimetics and the orally administered gliptins, and our study exclusively focusses on gliptins, and is not concerned with the GLP-1 mimetics data. The close relationship between chronic pancreatitis and pancreatic cancer is also a subject of intense research 46 . Another administrative database study of US adults with type 2 diabetes reported increased odds of hospitalization for acute pancreatitis for patients undergoing GLP-1 based therapies sitagliptin 13 . Once again, such correlation of GLP-1 based therapies to acute pancreatitis is contested by other studies 47 .
Our findings rationalize the elevated levels of serum lipase found in patients undergoing DPP4 inhibitor based therapies 28,29 , keeping in mind that other studies contradict these reports 30,31 . While several studies have reported that the GLP-1 mimetics do not induce pancreatitis in rats, mouse and/or monkey 48-50 , these studies did not include DPP4 inhibitors, which are the compounds that might be responsible for interactions with pancreatic proteins according to our study. It is to be noted however that these mimetics may have other physiological effects and 'the long-term consequences of sustained GLP-1 receptor activation in the human thyroid remain unknown and merit further investigation' 51 . Once again, the previous study 51 has been challenged by another group who note that 'findings previously reported in rodents may not apply to humans' 52 .
The orally administered gliptins differ in many aspects such as potency, excretion mechanism, target selectivity, half-life, metabolism and possible drug-drug interactions 9,53,54 . This difference is also highlighted in the different concentrations of vildagliptin and K579 that inhibit PI-PLC. A recent study has also noted the differential off-target inhibition of enzymes by vildagliptin and sitagliptin using a high-throughput, multiplexed assay 55 . Interestingly, the PI-PLC scaffold has a better match with the trypsin motif than with the DPP4 motif (Table 1). In order to be able to model these differences in our in silico search, it is important to be able to provide flexibility in the scoring mechanism.
To summarize, it has been noted in the case of GLP-1 based therapies that as 'evidence of harm accumulates, but is vigorously discounted' the 'burden of proof now rests with those who wish to convince us of their safety' 43 . Surveillance programs, real-life cohort studies and case-control studies can be supplemented by rational investigations of relevant proteins based on anecdotal reports 56 . The methodology proposed in the current work, which specifically demonstrates the effects of the DPP4 inhibitors, also presents a rational way of determining the inadvertent interactions of newly designed compounds with proteins, and thus prevent the recurrence of drug induced diseases being detected after considerable damage has already been inflicted on humans subjected to these drugs 57 .

Materials and methods
In silico analysis A comprehensive, non-redundant set of ~5000 human proteins (50% identity cutoff) was obtained from the PDB database 58 . The CLASP package (http://www.sanchak.com/clasp) used for querying these proteins using motifs from trypsin and DPP4 is written in Perl on Ubuntu 20 . Hardware requirements are modest -all results here are from a simple workstation (8GB ram), and runtimes for analyzing the ~5000 proteins was about 24 hours. Adaptive Poisson-Boltzmann Solver (APBS) and PDB2PQR packages were used to calculate the potential difference between the reactive atoms of the corresponding proteins 59,60 . The APBS parameters and electrostatic potential units were set as described previously in Chakraborty et al. 20 . All protein structures were rendered by PyMol (http://www. pymol.org/). Protein structures have been superimposed using MUSTANG 34 and DECAAF 35 .
Protein, substrate and reagents PI-PLC was purchased from Sigma. Vildagliptin (LAF-237) was obtained from Selleckchem, and K579 was obtained from Santa Cruz.

PI-PLC assay and inhibition using DPP4 inhibitors
Vesicle preparation and characterization. The appropriate lipids were mixed in organic solution, and the solvent was evaporated to dryness under N 2 . Solvent traces were removed by evacuating the lipids for at least 2 hours. The lipids were then swollen in 10 mM Hepes, 150 mM NaCl, pH 7.5 buffer. Large unilamellar vesicles (LUV) were prepared from the swollen lipids by extrusion and sized by using 0.1 µm poresize Nuclepore filters, as described by Ahyayauch et al. 36 . LUV composition was egg phosphatidylcholine: egg phosphatidylethanolamine: cholesterol at a 2:1:1 mole ratio. The average size of LUV was measured by quasi-elastic light scattering, using a Malvern Zeta-sizer instrument. Lipid concentration, determined by phosphate analysis, was 0.3 mM in all experiments.
Aggregation Assay. Enzyme activity was assayed measuring enzyme-induced vesicle aggregation. All assays were carried out at 39°C with continuous stirring, in 10 mM Hepes, 150 mM NaCl buffer (pH 7.5), in the presence of 0.1% BSA for optimum catalytic activity. Enzyme concentration was 0.16 U/mL, and liposomal concentration was 0.3 mM. Lipid aggregation was monitored in a Cary Varian UV-vesicle spectrometer as an increase in turbidity (absorbance at 450 nm) of the sample, as described by Villar et al. 37 . The data are average values of two closely similar experiments.

Analyzing known DPP4 inhibitors with solved structures.
In order to obtain all known structures of DPP4 with inhibitors bound to the active site, we did a search for the keyword dipeptidyl-peptidase on the PDB database, and choose proteins with DPP4 inhibitors as ligands. There are 76 such unique compounds (defined by three letter codes) that are reported to date (May 2014). We docked the DPP4 inhibitor to the PIPLC active site using DOCLASP 40 .

Data availability
figshare: Phosphoinositide-specific phospholipase C inhibition data using the dipeptidyl peptidase-IV inhibitors K-579 and LAF-237, http://dx.doi.org/10.6084/m9.figshare.880620 Author contributions SC, ARR and BA performed the experiments. All authors analyzed the data, and contributed equally to the writing and subsequent refinement of the manuscript.

Competing interests
No competing interests were disclosed.   The primary issue now is the speculation in the title.

Grant information
The title seeks to extrapolate the obtained data on two compounds to suggest that it is applicable to all DPP-IV inhibitors.
That is, the speculation of this paper is that the presented data is relevant to an entire drug class. The comments and the title should be restricted to one or two of the compounds that were studied in this paper. Moreover, K-579 is not a diabetes drug. In this context, the title needs changing to avoid ambiguity. I suggest: "The dipeptidyl peptidase IV inhibitor vildagliptin used in type 2 diabetes inhibits a phospholipase C: a case of promiscuous scaffolds in proteins." or "The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins." This study complements the much broader work using focused, direct technology for measuring and detecting off-target inhibition. That paper is published in Nature Chemical Biology in 2014 ( ). That study similarly showed that vildagliptin inhibits enzymes other than Bachovchin 2014 et al. DPP-IV. That study showed that DPP4 inhibitors differ, such that sitagliptin does not inhibit other enzymes.
The authors need to comment and restrict their conclusions to the compounds that they studied rather than imply that DPP-IV inhibiting compounds that they did not study, such as sitagliptin, have similar characteristics to the compounds that they did study.
The data of this study is biochemical yet 16 of the cited references concern the safety of DPP-IV 4.
The data of this study is biochemical yet 16 of the cited references concern the safety of DPP-IV inhibition. The manuscript now carefully does not draw a link to drug safety; the title needs to do the same.
As the paper is focused upon DPP-IV structure and function, more papers on this topic could be cited and linked with the data. For example, the author's amendment mentions contacts in DPP-IV at Glu205, Glu206 and Tyr662. The authors could state that Glu205 and Glu206 have been shown to be essential for catalysis by DPP-IV and cite the paper . Abbott (1999)  We would like to thank you for your positive comments, and your informative suggestions.
We agree with your suggested change in the title. In the latest version, we have also cited the research you have brought to our attention. The successful targeting of DPP4 using small molecule compounds to treat type 2 diabetes has attracted a great deal of attention towards the study of this protease.
The authors applied sophisticated techniques that they have developed in order to discover that two DPP4 inhibitors, including one that is in limited clinical use, can to some extent inhibit the activity of a bacterial lipase (PI-PLC). Many lipases and esterases and hydrolases including DPP4 and related enzymes use the hydrolase fold and the authors show how this related protein topology can alpha/beta place the residues in positions that are sufficiently similar to interact with an inhibitor.
The major difficulty with this paper is that it attempts to connect these data with possible clinical outcomes. No evidence for such a link is presented. Therefore, the title and much of the conclusions need to be modified so that they reflect the data without speculation.
Two inhibitors of DPP4, LAF237 and K-579, were studied. K-579 is not in clinical use. LAF237 is licensed in Europe and is known to exhibit some inhibition of the DPP4-related proteases DPP8 and DPP9. The extent of inhibition of DPP8 and DPP9 by LAF237 is believed not to have physiological effects in humans. The IC50 of LAF237 on DPP9 is less than 0.01 mM. The IC50 of LAF237 on bacterial PI-PLC is 0.1mM, which is close to the lower limit of detection of inhibition of an enzyme. No mammalian homolog of PI-PLC was examined.
The literature that the authors cite to suggest that DPP4 inhibition might be detrimental for human health, particularly the pancreas, is data on sitagliptin or exenatide. Exenatide is not a DPP4 inhibitor and sitagliptin is quite different to LAF237, both in protease specificity and in chemical structure. The contact points of LAF237 and sitagliptin in the catalytic site of DPP4 differ considerably. The authors present no data on sitagliptin or any other DPP4 inhibitor (other than LAF237) that in is the clinic.
The images of overlaid catalytic triads of various enzymes presented in Fig 1 and Fig 3 need to be depicted in 3D in order to evaluate how close they are in 3D. Intermolecular distances should be shown on thee figures. To convince the reader that LAF237 sits into and makes contacts with enzymes other than DPP4, we need to see the compound docked into the structure of each enzyme of interest.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.

Competing Interests:
Author Response 10 Dec 2014 , Tata Institute of Fundamental Research, India

Sandeep Chakraborty
We would like to thank you for taking the time to review this paper, and also for your insightful comments. We also apologize for the inordinate time taken to respond to your comments. A lot of this time was spent in understanding docking methods, instead of blindly applying this to the problem at hand. A by-product of this learning process was the implementation of a new method (DOCLASP) for docking molecules to proteins . We have docked vildagliptin to the PI-PLC structure complexed with myo-inositol using DOCLASP. Based on your suggestion, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date.
Please find out detailed responses to your comments below.
The successful targeting of DPP4 using small molecule compounds to treat type 2 diabetes has attracted a great deal of attention towards the study of this protease. The authors applied sophisticated techniques that they have developed in order to discover that two DPP4 inhibitors, including one that is in limited clinical use, can to some extent inhibit the activity of a bacterial lipase (PI-PLC). Many lipases and esterases and hydrolases including DPP4 and related enzymes use the alpha/beta hydrolase fold and the authors show how this related protein topology can place the residues in positions that are sufficiently similar to interact with an inhibitor. The major difficulty with this paper is that it attempts to connect these data with possible clinical outcomes. No evidence for such a link is presented. Therefore, the title and much of the conclusions need to be modified so that they reflect the data without speculation.
We have tried to keep away from taking sides on the clinical outcomes, since that is not our 1 We have tried to keep away from taking sides on the clinical outcomes, since that is not our forte. Also, we believe our title is innocuous in that context -it just speaks of promiscuous scaffolds. We only highlight that if (and only if) our data of PIPLC inhibition holds true for human lipases, then it might provide some arguing points for those worried about the side effects of these drugs.
For example, we say 'The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here'. If you could kindly point out specifically any speculations that is unwarranted, we will modify those. Since this study does not emphasize on the clinical relevance of the inhibitions (but on the methodology of finding such interactions), and we are not a group specializing in diabetes, we believe the choice of the inhibitors would not alter our reasoning our conclusions.
The IC50 of LAF237 on DPP9 is less than 0.01 mM. The IC50 of LAF237 on bacterial PI-PLC is 0.1mM, which is close to the lower limit of detection of inhibition of an enzyme.
We agree to this point. However, K-579 was inhibiting even at nanomolar concentrations.

No mammalian homolog of PI-PLC was examined.
We are currently evaluating that possibility.
The literature that the authors cite to suggest that DPP4 inhibition might be detrimental for human health, particularly the pancreas, is data on sitagliptin or exenatide. Exenatide is not a DPP4 inhibitor and sitagliptin is quite different to LAF237, both in protease specificity and in chemical structure.
We were referring to the inhibitor part of the data, but that point needs to be made explicit as you have correctly pointed out. Also, we agree that the possible difference of sitagliptin with LAF237 needs to be stated. We have modified the text to include these criticisms. Once again, we reiterate we intend not to comment on clinical outcomes or debates, but to suggest a rational methodology to act as a guide for tests that look for possible interactions.
The contact points of LAF237 and sitagliptin in the catalytic site of DPP4 differ considerably. The authors present no data on sitagliptin or any other DPP4 inhibitor (other than LAF237) that in is the clinic.
We have included a comprehensive study on the contact points of various inhibitors. Once again, this does not negate any of our conclusions.
The images of overlaid catalytic triads of various enzymes presented in Fig 1 and Fig 3 need to be depicted in 3D in order to evaluate how close they are in 3D.
The 3D images of the superimposition of these enzymes are not pleasing to the eye, since 1.
The 3D images of the superimposition of these enzymes are not pleasing to the eye, since they lack structural homology. However, we have added a PyMol script in case someone wishes to do that (Superimposeproteins.p1m). The script specifies the color coding of the residues.
Intermolecular distances should be shown on thee figures.
Once again, we think that the intermolecular distances clutter the figure. The superimposition gives an approximate idea of the congruence. The exact values are specified in Table 1. We have modified the legend of Fig.3 to specify that.
To convince the reader that LAF237 sits into and makes contacts with enzymes other than DPP4, we need to see the compound docked into the structure of each enzyme of interest.
As mentioned previously, we have docked sitagliptin to PI-PLC using DOCLASP . We have provided the Pymol script as supplementary data to help visualize the docking. There is no solved structure where LAF237 inhibits DPP4.
Once again, we are thankful for the comments. We hope that we have addressed your concerns by the changes that we have made, and that the manuscript will be found suitable in the modified form.
Prediction (CLASP), that compares structural and charge similarities of catalytic sites to identify functionally similar proteins. This methodology was used to assess the potential for adverse events based on off target effects of the inhibitors of DPP-IV. Using CLASP, the authors had previously indentified a Bacillus cerus phosphoinositide specific phospholipase-C (PI-PLC) as similar in active catalytic site to the enzyme, DPP-IV. They used laboratory techniques to verify this finding.
In the present study, the authors demonstrated the ability of two separate DPP-IV inhibitors to significantly reduce the activity of this PI-PLC in the lab. Subsequent to this experimentation, the authors returned CLASP to identify catalytic sites in other proteins that might also be inhibited by DPP-IV inhibitors thereby yielding unforeseen inhibition and biological effects. As applied to the case of DPP-IV inhibitors, which are not extremely specific, the authors identify a number of other proteins that could be promiscuously impacted by DPP-IV inhibitors thereby providing mechanisms for unexpected adverse events. Although the significance of DPP-IV inhibitor related adverse events has yet to be determined, the fact that changes have been reported non-clinically and clinically are undeniable. Eventually, the benefit of these molecules may far outweigh their associated risks, but the authors provide a potential path forward for investigation of unexpected events with this class of drug. If contradictory reports persist, this path may require further illumination.
The approach is theoretically similar to using structural similarities to identify off target receptor binding and consequent biological effects, an expanding approach in safety assessment and in identification of mechanisms for adverse events in the pharmaceutical lifecycle. Similarly, this method could be predictive for off target effects and suggest what those effects might be. However, whether this is a method that can be generally applicable to other molecules is beyond my ability to comment and the scope of this work. We would like to thank you for taking the time and reviewing our paper. Your positive comments encourage us to further our research in this area.
We concur with your statement -"Eventually, the benefit of these molecules may far outweigh their . And it is our endeavor to improve the accuracy and generality of our method associated risks" through different compounds. We would specifically like to highlight another case of antagonist binding identified through CLASP, although in this case most alkaline phosphatases were not affected -Chakraborty (2012) et al.
The data for PI-PLC inhibition using DPP4 inhibitors, as shown in Figure 2, are average values of two closely similar experiments. We will revise the manuscript to include this point when we hear from another referee.