On 2014 Feb 23, Ferenc Zsila commented:

Although the tocainide analogues studied here bear an asymmetric center (see in Fig. 1), their stereochemical characterization is missing. It remains unclear whether racemic or optically pure samples were used for the binding experiments.

Due to the presence of the chiral carbon atom, enantiomers of these molecules display CD activity below 300 nm which may overlap with the induced CD signals of the RSA-bound site markers used in the study (see the induced CD curves of diazepam, phenylbutazone, and ketoprofen in Fig. 6-9). Such a mutual spectral perturbation makes the reliable interpretation of the CD displacement results difficult. Unfortunately, CD spectra of free and albumin-bound forms of the enantiopure tocainide analogues are not presented.

The use of salicylate as the marker of Sudlow's site I (subdomain IIA) is ambiguous. It is a tiny molecule which can associate to multiple sites as demontrated by crystallographic results which revealed at least three, multidomain binding positions of diiodosalicylic acid on fatty acid-free bovine and equine serum albumin (Sekula B, 2013). Furthermore, the drug binding cavity of subdomain IIA is large enough to simultaneously accomodate two ligand molecules (Ghuman J, 2005). Thus, tocainide and its derivatives may co-bind with salicylate to this site. The same is hold for the case of phenylbutazone.

According to the Experimental Section, fatty acid-free RSA was used in all experiments. It is proposed that the primary binding site of tocainide analogues is located in subdomain IIIA which hosts one of the three highest-affinity binding sites of dietary fatty acids. Since fatty acids are the most abundant physiological ligands of serum albumin (Simard JR, 2006), fatty acid displacement measurements should also been performed to obtain more realistic data on the RSA binding behaviour of tocainide analogues.

In the course of mapping potential binding sites, the authors focuses exclusively on the classical Sudlow sites located in subdomain IIA and IIIA, respectively. Thus, they completely ignore recent findings showing the existence of a third primary drug binding area within subdomain IB (Zsila F, 2013). Biliverdin is the specific CD marker of this site which could be used for testing the subdomain IB binding of tocainide derivatives. This would be important since X-ray crystallographic studies verified the accomodation of the structurally related lidocaine molecule at the open entrance of the large binding crevice in subdomain IB (Hein KL, 2010).

Neither RSA nor HSA affinity constants of the tocainide analogues are reported in the paper. The number of the binding sites is also missing.

As it is claimed in the Conclusions, the tocainide analogues "showed ... a competitive behaviour with diazepam and bilirubin". Taking into consideration the very high-affinity RSA binding of bilirubin (Ka = 2-4 x 10⁶ M^-1 ) compared to the weak RSA association of the analytes (Ka ~ 10⁴ M^-1 ) this postulation is rather questionable. Additionally, in a recent work the primary RSA binding site of bilirubin has been assigned to subdomain IIA (Goncharova I, 2013), which was excluded by the present study as a possible binding locus of tocainides: "The addition of increasing concentrations of compounds BO3 and BO14 up to a molar ratio [competitor]/[marker] 8/1 determined a blue-shift of the induced CD spectrum of [RSA]/[PBU] 1/1 complex (Fig. 6). This result suggests an allosteric interaction between the site I marker and competitors, without a significant displacement of the marker, consistently to the results obtained by affinity chromatography." (p. 9).

On 2014 Feb 23, Ferenc Zsila commented:

Although the tocainide analogues studied here bear an asymmetric center (see in Fig. 1), their stereochemical characterization is missing. It remains unclear whether racemic or optically pure samples were used for the binding experiments.

Due to the presence of the chiral carbon atom, enantiomers of these molecules display CD activity below 300 nm which may overlap with the induced CD signals of the RSA-bound site markers used in the study (see the induced CD curves of diazepam, phenylbutazone, and ketoprofen in Fig. 6-9). Such a mutual spectral perturbation makes the reliable interpretation of the CD displacement results difficult. Unfortunately, CD spectra of free and albumin-bound forms of the enantiopure tocainide analogues are not presented.

The use of salicylate as the marker of Sudlow's site I (subdomain IIA) is ambiguous. It is a tiny molecule which can associate to multiple sites as demontrated by crystallographic results which revealed at least three, multidomain binding positions of diiodosalicylic acid on fatty acid-free bovine and equine serum albumin (Sekula B, 2013). Furthermore, the drug binding cavity of subdomain IIA is large enough to simultaneously accomodate two ligand molecules (Ghuman J, 2005). Thus, tocainide and its derivatives may co-bind with salicylate to this site. The same is hold for the case of phenylbutazone.

According to the Experimental Section, fatty acid-free RSA was used in all experiments. It is proposed that the primary binding site of tocainide analogues is located in subdomain IIIA which hosts one of the three highest-affinity binding sites of dietary fatty acids. Since fatty acids are the most abundant physiological ligands of serum albumin (Simard JR, 2006), fatty acid displacement measurements should also been performed to obtain more realistic data on the RSA binding behaviour of tocainide analogues.

In the course of mapping potential binding sites, the authors focuses exclusively on the classical Sudlow sites located in subdomain IIA and IIIA, respectively. Thus, they completely ignore recent findings showing the existence of a third primary drug binding area within subdomain IB (Zsila F, 2013). Biliverdin is the specific CD marker of this site which could be used for testing the subdomain IB binding of tocainide derivatives. This would be important since X-ray crystallographic studies verified the accomodation of the structurally related lidocaine molecule at the open entrance of the large binding crevice in subdomain IB (Hein KL, 2010).

Neither RSA nor HSA affinity constants of the tocainide analogues are reported in the paper. The number of the binding sites is also missing.

As it is claimed in the Conclusions, the tocainide analogues "showed ... a competitive behaviour with diazepam and bilirubin". Taking into consideration the very high-affinity RSA binding of bilirubin (Ka = 2-4 x 10⁶ M^-1 ) compared to the weak RSA association of the analytes (Ka ~ 10⁴ M^-1 ) this postulation is rather questionable. Additionally, in a recent work the primary RSA binding site of bilirubin has been assigned to subdomain IIA (Goncharova I, 2013), which was excluded by the present study as a possible binding locus of tocainides: "The addition of increasing concentrations of compounds BO3 and BO14 up to a molar ratio [competitor]/[marker] 8/1 determined a blue-shift of the induced CD spectrum of [RSA]/[PBU] 1/1 complex (Fig. 6). This result suggests an allosteric interaction between the site I marker and competitors, without a significant displacement of the marker, consistently to the results obtained by affinity chromatography." (p. 9).