The Effect of Thiol Containing Compounds on the Kinetics of Sickle Hemoglobin Polymerization 

Review Article

The Effect of Thiol Containing Compounds on the Kinetics of Sickle Hemoglobin Polymerization 

Corresponding author:  Dr. Ahmed S. Mehanna, MCPHS University, School of Pharmacy, Department of Pharmaceutical Sciences 179 Longwood Avenue, Boston Ma 02115, Tel: 617-732-2955;
Abstract
The current research reports the anti-polymerization activities of thirteen thiol containing compounds. The evaluation was conducted using our newly reported high throughput kinetic assay designed to study sickle hemoglobin polymerization kinetics. The evaluated compounds ranged from thiol containing amino acids and chemicals to marketed drugs containing the thiol functionality. The evaluated compounds/drugs included, thiosalcylic acid, captopril, methimazole,6-mercaptopurine,cysteine, mercaptosuccinicacid, meso-2,3-dimercaptosuccinic acid, N-acetyl-L-cysteine, L-glutathione, 2-mercaptopyridiene-3-carboxylic acid, Mesna, and D&L-penicillamine. Most of the evaluated compounds exhibited a concentration-dependent delay in sickle hemoglobin polymerization at low concentration range of 0.2-2.0 mM. Thiol containing drugs included in the study that showed significant delay on polymerization are promising for potential second use indication to treat sickle cell anemia.
   Keywords: Sickle hemoglobin; Sickle Cell Anemia; Thiol Compounds; Polymerization Inhibitors
Introduction
In Search of drugs that may be potentially useful to treat sickle cell disease, we recently developed a fast and economic high throughput assay to evaluate the effects of organic compound on sickle hemoglobin (HbS) polymerization kinetics. [1] through the development of the assay, we have discovered that thiosalcylic acid (TSA) showed very promising delay in HbS polymerization. Delaying the initial polymerization has a very important significance out come in reducing the sickle cell crises. Deoxygenated blood can take up to 14.5 seconds in the circulation before being re-oxygenated, so therapeutic effect could be achieved by compounds that would cause either considerable delay for the onset of polymerization, or reduction in the rates of polymerization in the first 50% of the progression curve. [2] The finding that TSA showed high activity in delaying the polymerization process; prompted us to investigate the potential effects of additional thiol containing compounds on the kinetics of HbS polymerization. The selected thiol compounds ranged from drugs already in clinical use for different purposes to amino acids and metal chelating agents. The investigated compounds included, in addition to TSA as a reference, Captopril (antihypertensive drug), Methimazole (anti-thyroid drug), 6-mercaptopurine (anticancer), cysteine (amino acid), N- acetyl-L-cysteine, mercaptosuccinic acid, meso-2,3-dimercaptosuccinic acid, D-penicillamine, L-penicillamine, the (metal chelators agents), mesna (antidote drug), L-glutathione (endogenous bio-molecule) , and 2-mercaptopyridiene- 3-carboxylic acid (pyridine analog of thiosalcylic acid. The structures and abbreviations of all tested compounds are shown in Figure 1. The polymerization of the HbS is produced through a double nucleation mechanisms; homogeneous and heterogeneous nucleation. Each is followed by a rapid stage of fiber growth. [3] The polymerization kinetics can be studied by monitoring the effect of the test compound on the delay time in the start of polymerization. Any prolongation of the delay time is clinically significant in reducing the sickle cell crises resulting from the blood vessels occlusion caused by distorted cell shape, which in turn results from the polymerization of HbS inside the red cell. Our approach is to test and find molecules capable of prolonging the delay time for even few  seconds to allow safe passage of sickle red blood cells to pass through the micro-vascular bed before it deformation in the low oxygen tension region. Delaying the nucleation for as just few second is enough to prevent cell distortion and allow it to cross through the vascular system without occlusion. [4] All selected thiol compounds were evaluated for their effects on HbS polymerization kinetics using our recently reported high throughput assay. [1]
Figure 1. Thiol containing compounds and drugs tested in the study.
Material and methods

All reagents and drugs were purchased from Sigma Aldrich. The assay was run in high potassium phosphate buffer (1.5M), in which of the assay components were dissolved in, including,compound/drug under testing, sickle hemoglobin, the deoxygenating
agent 1.0 M potassium metabisulfite. Biotek Synergy HT Plate reader was programmed with Gen5 software to maintain the temperature at 37oC before and throughout the kinetic analysis and gently shake the samples for 3 seconds before the first reading. In the assay, 3 groups were used: A blank group as a singlet in which 20μl of the buffer was dispensed to the wells, a control group as a triplet in which 10μl buffer was added to 10μl of the HbS solution, and the sample group as quadruplets of each concentration of the potential agent in which 10μl of each concentration of the potential agents were added to 10μl of the HbS solution. Prior to setting the machine to start the kinetic analysis, 20μl of the potassium metabisulfite solution was added to each well including the blank. The polymerization of HbS was monitored by measuring the optical density at 700nm for 20 minutes. In each run, the optical density readings were taken at 41 time points that are 30 seconds apart. For each drug, the experiment was repeated 4 times, and the average of the optical density values for the control and each compound concentration were plotted against time using GraphPad Prism software.The anti-polymerization effects of the screened agents in our study were assessed by comparing the times taken by the control and different drug concentrations to achieve optical density values equivalent to 10% and 50% of the control’s maximum polymerization ( TC10 and TC50 are designated for the control group, while TD10 and TD50 are designated for the tested compounds/drugs group). To obtain the values of TC10, TC50, TD10 and TD50, Graphpad was programmed to create a pointto- point curve, calculate the optical density values every 0.02 seconds, and interpolate the optical density values equivalent to 10% and 50% of the control’s maximum. In the point-topoint curve, the software creates a series of line segments between data points and calculates the values of optical density between them. The values of TD10 and TC10were compared to each other, while the values of TD50and TC50were compared to each other using One-way ANOVA analysis. In cases when ANOVA showed a significant difference, Tukey’s post-hoc analysis was used to compare the values of the control and different concentrations of each drug. The values of TC10, TC50, TD10 and TD50 obtained with each drug were represented in tables as mean of 4 experiments, and the dispersion was expressed as standard deviation from the mean. The level of significance was represented in front of the values by asterisks. Finally, the delay times of each drug were represented graphically with asterisks that represents the level of statistical significance either from the delay times of the control or from the first significant delay time of a lower concentration. This method of data analysis was used some produced curves were not sigmoidal. Hofrichter and Eaton demonstrated that the time required to achieve 10% polymerization represents the rate of homogeneous nucleation.5 Based on that, the effect of different drugs on the nucleation rate will be compared by the time required to reach an equivalent of 10% polymerization of the control’s maximum (TC10) which represent both homogeneous nucleation and heterogeneous nucleation, and on the fiber growth rate by the values of TC50. It must be reiterated that deoxygenated blood can take up to 14.5 seconds in the circulation before being re-oxygenated, so therapeutic effect could be achieved by compounds that would cause either considerable delay for the onset of polymerization, or reduction in the rates of polymerization in the first 50% of the progression curve [2].Results and Discussion
The screened compounds/drugs are with one of the following structural features: either with a cyclic system or with no cyclic system. The compounds with cyclic system include TSA, 2-MPC, MMI, 6-MP, and captopril; while the compounds that do not have a cyclic system include MSA, MDMSA, L-Cysteine , NAC, L-GSH, mesna, D-Penicillamine, and L-Penicillamine. Another observation in the structures of the tested compounds is that, with the exception of 6-MP and 2-MMI, all tested compounds possessed an acidic functionality. The indicated structural features have variable effects on the anti-polymerization activities as detailed below. Results and discussion are addressed below for each individual screened compound with supporting graphs and tables for the T values, followed by the structure activity discussion.Thiosalcylic acid

Thiosalcylic acid (TSA) is an anti-inflammatory drug available in the market (Thiocyl®). TSA showed a concentration dependent anti-polymerization activity in a concentration range of 0.2-1.6 mM, figure2, with calculated TD50 of 2.7, 4.0, 7.7, and 14.5 minutes for 0.2mM, 0.4mM, 0.8mM, and 1.6mM respectively. The TD10 and TD50values are depicted in tables 1&2.

Figure 2. Effects of thiosalcylic acid on HbS polymerization.

Table 1. The effect of different TSA concentrations of TSA on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10orTD50/ TC50 of the control.

Table 2. The delay time caused by different concentrations of TSA expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Mercaptopyridine-3-carboxylic acid

The compound 2-Mercaptopyridine-3-carboxylic acid (2-MPC) was chosen for its structural similarity to TSA with a pyridine ring instead of benzene in TSA. Compared to TSA, 2-MPC showed dramatic loss in the activity. It showed much lower TD- 50values at much higher concentrations of 4.6 and 5.2 minutes for 110 and 210mM respectively (Figure 3 and tables 3&4).


Figure 3. Effect of 2-mercaptopyridine-3-carboxylic acid 2-mercaptopyridine- 3-carboxylic acid on HbS polymerization.

Table 3. The effect of different concentrations of 2-Mercaptopyridine- 3-carboxylic acid on the delay time of HbS polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001).The number of Folds of increase in the delay time = TD10/ TC10 orTD50/TC50.

Table 4. The delay time caused by different concentrations of 2-MPC expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Mercapto-1-methylimidazole (2-MMI)

2-Mercapto-1-methylimidazol, also known as methimazole, is an antithyroid drug available in the market under the nameTapazole®. The drug was chosen to test the effect of lacking the carboxylic group on activity. The drug showed no significantchange in the TD50values but a slight change in the TD- 10value. (Figure. 4 & Tables 5&6).

Figure 4. Effect of 2-mercapto-1-methylimidazole on HbS polymerization.

Table 5. The effect of different concentrations of 2-MMI on the delay times of sickle hemoglobin polymerization.The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10orTD50/TC50.

Table 6. The delay time caused by different concentrations of 2-MMI expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

6-Mercaptopurine (6-MP)

The drug is an immunosuppressive drug used to treat leukemia under the trade name of Purinethol®. As 2-MMI, 6-MP was chosen as another example of a commercially available thiol which lacks acidic functionality. The drug showed no significantchange in TD50values (Fig. 5 &Tables 7&8)

Figure 5. Effect of 6-MP on HbS polymerization.

Table 7. The effect of different concentrations of 6-MP on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10 and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD.Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001).The number of Folds of increase in the delay time = TD10/ TC10orTD50/TC50.

Table 8. The delay time caused by different concentrations of 6-MP expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Captopril

Captopril is a member of the ACE inhibitors antihypertensive class. It was thought to be another good candidate given its antialbuminuric and antihypertensive benefits to the sickle cell anemia patients.28,29 It showed anti-polymerization activitiessimilar in a large part to that of TSA with similar TD50 values of 6.4, 9.4, 11.2, 12.2, and 14.1 minutes at concentrations of 0.13mM, 0.25mM, 0.5mM, 1.0mM, and 2.0mM respectively. One slight difference between TSA and Captopril is that the lattershowed TD50 value of 14min at 2mM, which was caused by 1.6mM of TSA. (Fig. 6 & Tables 9&10).

Figure 6. Effect of Captopril on HbS polymerization.

Table 9. The effect of different concentrations of Captopril on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001).The number of Folds of increase in the delay time = TD10/ TC10orTD50/TC50.

Table 10. The delay time caused by different concentrations of Captopril expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Mercaptosuccinic acid (MSA)

The compound was chosen to for its character of not possess cyclic system. MSA is used to chelate gold and reduce its sideeffects in injections for the management of Rheumatoid Arthritis (Myocrisin ®). MSA showed moderate activity, with TD50values of 2.2, 2.8, 4.4, and 8.8 minutes at 0.19mM, 0.38mM, 0.75mM, and 1.5mM respectively. (Figure 7, tables 11&12).

Figure 7. Effect of mercaptosuccinic acid on HbS polymerization.

Table 11. The effect of different concentrations of MSA on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10orTD50/TC50.

Table 12. The delay time caused by different concentrations of MSA expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Meso-2, 3-dimercaptosuccinic acid (MDMSA)

MDMSA is a drug used to treat lead poisoning (Chemet®). The structure is characterized by being with two thiol groups.MDMSA showed weak activity that does not appear to be concentration dependent since 2.0mM and 32 mM concentrations are almost overlapping with very little differences in the TD50 (Fig 8 & Tables 13&14).


Figure 8. Effect of meso-2,3-dimercaptosuccinic acid on HbS polymerization.

Table 13. The effect of different concentrations of MDMSA on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD.Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10 or TD50/ TC50.

Table 14. The delay time caused by different concentrations of MDMSA expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

L-Cysteine

Cysteine; being an amino acid, differs from the above compounds in having an amino functional group. Interestingly, it exhibited the highest activity among all of the tested compounds withTD50 was more than 20min. It also showed an extensivedelay to the onset of polymerization as reflected by the TD10values (Fig. 9&Tables 15&16).

Figure 9. Effect of L-Cysteine on HbS polymerization.

Table 15. The effect of different concentrations of L-Cysteine on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001).The number of Folds of increase in the delay time 10/ TC10 orTD50/ TD50.

Table 16. The delay time caused by different concentrations of L-Cysteine expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

N-acetyl-L-cysteine

N-acetyl-L-cysteine (NAC) is a mucolytic drug (Acetylcysteine ®) was chosen to test the importance of the basic amino groupof cysteine on activity. NAC also showed much lower activity  than L-Cysteine. with TD50 values of 7.5, 8.9, 10.1, 11.3 minutes,at 0.25mM, 0.5mM, 1.0mM, and 2.0mM respectively (Fig. 10 and tables 17&18).

Figure 10. Effect of N-acetyl-L-cysteine on HbS polymerization.

Table 17. The effect of different concentrations of NAC on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD.Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10or TD50/TC50.

Table 18. The delay time caused by different concentrations of NAC expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

L-Glutathione

L-Glutathione (L-GSH) was chosen for being a tripeptide with Cysteine component. L-GSH showed less antigelling activity than L-Cysteine (TD50 values are 7.8, 9.3, 10.8, 12.3, 14.5 minutes at 0.25, 0.5, 1.0, 2.0, and 4.0mM, respectively (Figure 11 and tables 19&20).

Figure 11. Effect of L-Glutathione on HbS polymerization.

Table 19. The effect of different concentrations of L-GSH on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD.Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10or TD50/TC50.

Table 20. The delay time caused by different concentrations of L-GSH expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

D-Penicillamine

D-Penicillamine is a drug used to treat Wilson’s disease and Rheumatoid arthritis (Cuprimine ®). D-Penicillamine was chosen for its structural similarity to Cysteine. It has two additional methyl groups on the β-carbon of Cysteine. D-Penicillamineshowed less activity than L-cysteine (TD50 values are 4.5, 5.8, 6.9, 9.7, and 10.5 minutes at 0.25mM, 0.50mM, 1.0mM, 2.0mM, and 4.0mM respectively (Figure 12 and Tables21&22).

Figure 12. Effect of D-Penicillamine on HbS polymerization.

Table 21. The effect of different concentrations of D-Penicillamine on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD. Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10or TD50/TC50.

Table 22. The delay time caused by different concentrations of D-Penicillamine expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

L-Penicillamine

As an enantiomer of the D-Penicillamine, it was chosen for screening to test the effect of stereochmistry, if any, on activity. L-Penicillamine showed similar antigelling activity to D- Penicillamine with TD50 values of 6.2, 6.9, 7.8, 10.0, and 11.6 minutes at 0.25mM, 0.50mM,1.0mM, 2.0mM, and 4.0mM respectively (Figure 13 and Tables 23 and 24).

Figure 13. Effect of L-penicillamine on HbS polymerization.

Table 23. The effect of different concentrations of L-Penicillamine on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD.Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10 orTD50/TC50.

Table 24. The delay time caused by different concentrations of L-Penicillamine expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Mesna

Mesna is a drug used to prevent hemorrhagic cystitis caused by some chemotherapeutic agents (Mesnex ®). Mesna, has theacidic functionality as sulfonate group and was chosen to assess if the sulfonic acid group can replace the carboxylic group seen in the above compounds and drugs. Mesna showed similar activity to mercaptosuccinic acid (MSA) with TD50 values of 5.7, 7.9, 9.6 and 10.7 at 0.13mM, 0.25mM, 0.5.0mM, and 1.0Mm respectively (Figure 14 and Tables 25&26).


Figure 14. Effect of mesna on HbS polymerization.

Table 25. The effect of different concentrations of Mesna on the delay times of sickle hemoglobin polymerization.

The times taken to achieve optical density values equivalent to 10% and 50% of the control’s maximum are designated TC10and TC50 for the control, and TD10 and TD50 for the drug groups. Values of TC10 and TC50 represent the mean of four runs ± SD.Asterisks represent the level of statistical significance compared to the control (*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001). The number of Folds of increase in the delay time = TD10/ TC10 or TD50/TC50.

Table 26. The delay time caused by different concentrations of Mesna expressed in T50.

T50 is the time taken to achieve 50% progression of polymer growth for each curve. T50 is expressed as mean of four runs ± SD.

Structure activity relationships

The activity of the 13 thiol compounds as inhibitors for HbS polymerization is summarized in table 27. And the same data is depicted in graphical for in figures 14,15 &16.

Table 27. Summary of the delay time range achieved by each compound with the concentration range of activity.

Figure 15. Graphical representation of the increase in the values of TD10 and TD50from the control values (TC10 and TC50) in TSA, 6-MP, 2-MPC, Captopril, 2-MMI, and L-GSH at various concentrations.

The Asterisks represent the degree of statistical significance from the control(*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001), while plus symbols represent the level of significance from the first significant lower concentration (+P<0.05,++P<0.01, +++P<0.001, and ++++P<0.0001).

Figure 16. Graphical representation of the increase in the values of TD10 and TD50from the control values (TC10 and TC50)inMSA,MDMSA, L-Cysteine, N-acetyl-L-cysteine, D- and L-Penicillamine at various concentrations.The Asterisks represent the degree of statistical significance from the control(*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001), while plus symbols represent the level of significance from the first significant lower concentration(+P<0.05,++P<0.01, +++P<0.001, and ++++P<0.0001).

Figure 17. Graphical representation of the increase in the values of TD10 and TD50from the control values (TC10 and T C50)in Mesna at various concentrations.The Asterisks represent the degree of statistical significance from the control(*P<0.05, **P<0.01, ***P<0.001, and****P<0.0001), while plus symbols represent the level of significance from the first significant lower concentration (+P<0.05,++P<0.01, +++P<0.001, and ++++P<0.0001).Among the cyclic thiol-containing compounds used in thisstudy, the highest anti polymerization activity was achieved with TSA and captopril which have similar activity at similar concentration ranges. On the other hand, 2-MPC, with its pyridine ring instead of the benzene ring in TSA, showed much lower activity at a very high concentration range (4.6 and 5.2 minutes for 110 and 210mM, respectively). Possible explanation is the nitrogen in the pyridine ring decreases the electron density over the ring and makes its partially polar and less lipophilic than the benzene ring. So this drop in activity could be because the binding site favors more lipophilic compounds, or compounds with negative charge density around the thiol group. Both 2-MMI and 6-MP did not show any activity. These were the only two compounds among the tested thiols thiol compounds that do not have acidic functionality; signifying that acidic functionality is a must for activity. Compounds with no cyclic system, such as MSA, showed TD50 values of 2.2, 2.8, 4.4, and 8.8 minutes at 0.19mM, 0.38mM, 0.75mM, and 1.5mM, respectively (table 27). MDMSA, another non-cyclic example with dithiol functionality, less activity when compared to MSA. MDMSA shows less delay times, possibly due to lower affinity to the binding site as result of the steric hindrance caused by the additional thiol group. On the other hand, mesna, with its sulfonate group two carbons apart from the thiol, had similar activity to MSA. The replacement of the carboxylate group with sulfonate group retained the activity. This suggests that the structural requirement for activity could be any acidic functionality and not only limited to the carboxylic group. The highest activity in this group of compounds was achieved with the amino acid L-cysteine which demonstrated its activity in 0.25-4.0 mM concentration range with more than 20 minutes delay at 1mM. D-penicillamine and L-penicillamine have similar structure to cysteine but differ in the presence of two methyl groups on the β-carbon. Both enantiomers showed moderate activity at concentration range of 0.25-4.0mM, with no difference between the two enantiomers. The addition of two methyl groups on the β-carbon of penicillamine greatly
decreased the activity probably due to the steric hindrance caused by these two methyl groups. This might explain why the two enantiomers did not show difference in their activity. NAC, acetylated version of cysteine, showed lower activity than L-cysteine (TD50 values of 7.5, 8.9, 10.1, 11.3 minutes, at 0.25mM, 0.50mM, 1.0mM, and 2.0mM of NAC respectively) This reduction in the activity could be attributed to the elimination of the positive charge on the amine group. Finally, L-GSH also showed less activity than L-cysteine. L-GSH possesses two carboxylic groups; however, the position of these groups is further away from the thiol functionality than cysteine.The effect of the screened thiol compounds on TD10 and TD50 were different as depicted in figures 14-16. MDMSA was the only compound that caused a significant delay in the TD50 values with no effect on the TC10, while other compounds (D-penicillamine, L-penicillamine, TSA, and captopril) caused a slight increase in the values of TC10. Moreover, L-cysteine which had the most activity caused extensive inhibition to both TD10 and TD50. The effect of these drugs on TD10 is suggested to be due to their interference with the nucleation processes.

For compounds which showed the longest delay in TD50 (L-cysteine, TSA and captopril), it is noteworthy that they alsoproduced the longest delay in TD10 among all the compounds tested. This highlights the influence of nucleation in the kineticsof HbS polymerization.

Conclusions

With the exception of 2-MPC and 6-MP; all studied thiols demonstrated anti-polymerization activities at concentration range of 0.2-4.0mM. The acidic functionality is essential for activity with variable strength varied depending on the structural features of each compound. Furthermore, substituting the carboxylic group with sulfonate retained the activity as seen in Mesna. The distance of the acidic functionality from the thiol group seems to influence the interfere more with polymerization. Compounds that have the thiol and carboxylic group are two carbons apart have optimum requirements for high activity as seen in cysteine, TSA, and mesna. The different effects of these compounds on TD10 are due to the difference in their inhibitory activity against the nucleation phases. The mechanism of interference of these compounds with the nucleation steps offers an interesting topic for future studies. Lastly, the study revealed anti polymerization activities for several drugs. Such drugs are good candidates for clinical evaluation to treat sickle cell anemia as second indication.

Acknowledgment

The authors are thankful for MCPHS University for funding this research

References

1. Ahmed S Mehanna. High Throughput Kinetic Assay for Screening Potential Inhibitors of Sickle Hemoglobin Polymerization. Med Chem (LOS Angeles), 2017, 7: 929-932.

2. Huang Z, Hearne L, Irby C E, King SB, Ballas SK et al. Kinetics of increased deformability of deoxygenated sickle cells upon oxygenation. Biophys. J. 2003, 85(4): 2374-2383.

3. Mirchev R, Ferrone FA. The structural link between polymerization and sickle cell disease. J. Mol. Biol. 1997, 265(5): 475-479.

4. Mozzarelli A, Hofrichter J, Eaton WA. Delay time of hemoglobin S polymerization prevents most cells from sickling in vivo. Science. 1987, 237(4814): 500-506.

5. Lottenberg R, Hassell KL. An evidence-based approach to the treatment of adults with sickle cell disease. Hematology Am. Soc. Hematol. Educ. Program. 2005, 58-65

Be the first to comment on "The Effect of Thiol Containing Compounds on the Kinetics of Sickle Hemoglobin Polymerization "

Leave a comment

Your email address will not be published.


*