Development of Selective (R)-Salsolinol N-Methyltransferase Inhibitors for Study and Treatment of Parkinson's Disease

This is a paper I wrote for a Biochemistry class while getting my undergraduate degree. It's a mock grant proposal, which means in real life you would write something similar when trying to get funding for whatever project you want to study. In this case, the class was largely about the design of small molecule inhibitors (eg. “drugs”), and I chose to apply what I had learned to the treatment of Parkinson's Disease. I'm releasing this draft under the standard Devtome CC license, so feel free to use or adapt it for actual research purposes - I'd love to see this idea become a reality.


The role of endogenous neurotoxins in Parkinson's Disease

Parkinson's Disease (PD) is the second most common neurodegenerative disease worldwide. About 10% of PD cases are familial, but the remaining 90% have no specific known cause. Due to the idiopathic nature of the disease, the majority of modern medical treatment for PD has been focused on management of disease symptoms, rather than treating the underlying cause. It has been shown that oxidative stress plays a key role in the dopaminergic neuron death that characterizes PD pathology (1). Reactive oxygen species (ROS) contribute to this pathology by causing lipid peroxidation, DNA damage, protein oxidation, and eventually leading to cell death via apoptosis (1,2). Environmental factors, specifically both exogenous and endogenous neurotoxins, have been hypothesized to play a major role in the etiology of idiopathic PD (4). Salsolinol, an endogenous neurotoxin, has been implicated in a number of degenerative diseases; most significantly, PD. Salsolinol and its derivatives have been shown to cause dopaminergic cell death through a ROS-activated cascade (4). N-methyl-salsolinol in particular is important in causing the pathology of PD (4,5,6). Modern approaches to drug development for PD have focused on neuroprotectant compounds, such as the antioxidant Trolox and the anti-apoptotic Rasagiline (1,2,3). While these compounds show a lot of promise in the treatment of PD and other neurodegenerative diseases, they do little towards the elucidation and treatment of the underlying cause of PD.

N-methyl-salsolinol transferase as a potential drug target

While the synthesis salsolinol itself seems like an attractive target, it's natural function is unclear, it may be important in natural neuromodulation and in some situations it may potentially play a reverse role a neuroprotectant (4). N-methyl-salsolinol biosynthesis, however, is much better suited for inhibition. N-methyl-salsolinol has no known function other than as a neurotoxin, and it has been directly implicated in the pathology of PD (4,5,7). N-methyl-salsolinol is synthesized from salsolinol by a neutral methyltransferase enzyme (4,7). This enzyme, (R)-Salsolinol N-Methyltransferase (SNMT), has increased activity in patients with PD (4,6,7). Although it has been shown that salsolinol can be used to induce PD in animal models, it is unknown whether salsolinol itself, or it's N-methylated derivative is the main cause of salsolinol pathology (4,8). Therefore, SNMT makes an attractive drug target for two reasons: First, N-methyl-salsolinol is an endogenous neurotoxin known to significantly contribute to PD, thus an SNMT inhibitor would be an effective treatment for PD, especially in conjunction with a neuroprotectant. Secondly, an SNMT inhibitor would be instrumental in elucidating whether salsolinol alone or its derivatives are the cause of salsolinol pathology.

Fragment Based Screening: a robust and effective approach to drug discovery

In contrast to High Throughput Screening (HTS), Fragment Based Screening (FBS) uses a small library of compounds with small molecular weights (~200 Da). Hits tend to bind targets efficiently, but with low affinity (10). Compounds with greater affinity can then be generated by overlapping hit compounds (9,10). There are several advantages to FBS in comparison to HTS, the most obvious being that the number of possible fragments that may bind a target is many orders of magnitude smaller than the number of possible compounds in HTS libraries (10). Thus, a small library of fragments may be able to traverse a larger chemical space than even the largest HTS libraries. Further, HTS libraries are optimized towards already known molecular mechanisms of inhibition, so FBS may be better suited towards finding compounds that bind novel targets (10). In fact, it has been shown that observed hit rates in FBS were roughly 10-1,000 times higher than conventional HTS techniques (10). Although HTS may be quicker to perform, it is often plagued by false positives, which complicates the process of lead optimization. In contrast, FBS is much less prone to artifacts due to its use of smaller molecules and more robust sampling techniques (10). Perhaps the most significant advantage of FBS is its ability to assist in structure-based drug design; in fact, the ability to produce potent inhibitors after lead optimization is as high as 93%, compared to 33% after HTS on the same targets (10).

Using Fragment Based Screening to create high affinity SNMT inhibitors

Although there has been no study of SNMT in regards to its inhibition, several inhibitors have been developed towards similar targets. In particular, FBS was used to develop several potent phenylethanolamine N-methyltransferase (PNMT) inhibitors (9). Even though there are many reasons to use FBS to find SNMT inhibitors, there are also several potential pitfalls that must be taken into account. One challenge is measuring the activity of hits with such weak binding affinities. One effective method that has been used is isothermal titration calorimetry (ITC) (9). A method to identify hits must also be selected. FBS by X-Ray crystallography (FBS-X) is very attractive, because it directly reveals the relationship between fragment structure and binding, which assists greatly in lead optimization (9). An obvious pitfall in this case is the potential difficulty of crystallizing SNMT. SNMT acts on two substrates, (R)-salsolinol and S-adenosyl-L-methionine (SAM) (**Figure 1**).

**Figure 1**. The reaction pathway of N-methylsalsolinol from salsolinol catalyzed by SNMT in the presence of SAM (11).

This is a potential problem because SNMT may not be able to be crystallized without the presence of SAM, in which case it would be a non-ideal FBS-X target. However, a similar challenge was overcome with PNMT, which does not readily crystallize without it's cofactor (9). Rapid soaking of the fragment in SNMT-SAM crystals is a possible solution to this problem. If crystallization proves unfeasible, there are a few alternative methods of hit characterization that could be used, in particular, MS or NMR (9,10).

Research Design and Specific Aims

In this study, we will develop and characterize high-affinity Salsolinol N-methyltransferase inhibitors through fragment-based X-ray crystallographic screening for use in the study and treatment of Parkinson's Disease.

Specific Aim 1: Generate a series of hit fragments that bind to SNMT via FBS-X

A small library of fragments will be purchased from ActiveSight, similar to the library used to develop PNMT inhibitors (9). We will then isolate and purify SNMT as described in (7). FBS will be conducted by X-ray crystallography. First, crystals of each fragment, SNMT, and SAM will be grown using rapid soaking methods. Next, the crystals will be flash-cooled and X-ray diffraction data will be gathered. Structures for each fragment will be solved using MIfit from ActiveSight (9). Active-site density will be assessed individually to determine presence or absence of ligand. Ligands shown to be present will be modeled and refined with COOT, PRODRG, and PHENIX as described in (9).

Specific Aim 2: Construction of a high-affinity SNMT inhibitor

Compounds found to bind to SNMT by FBS-X will subsequently be analyzed by ITC. The association constant (Ka; 1/Kd), enthalpy (ΔH) and stoichiometry (N) will be calculated using a single-site binding model. From the Ka and ΔH values, ΔG and ΔS can be calculated to determine the overall contributions of both enthalpy and entropy to binding. Enthalpically favorable hits that bind to different parts of the active site will be selected to develop and optimize several lead compounds. These selected fragments will be superimposed to construct several lead compounds, which will subsequently be characterized by X-ray crystallography and ITC in the same way the fragments were. Leads composed of several fragments will have much higher affinities than the fragments that they were composed from.

Specific Aim 3: Functional characterization of SNMT inhibitory compounds

Finally, the ability of each compound to inhibit SNMT in rat lymphocytes will be assessed using liquid chromatography coupled with electrospray time-of-flight mass spectrometry (11). The standard 6-hydroxydopamine (6-OHDA) lesion model of PD in rats may prove problematic, in that 6-OHDA may mimic the natural cause of PD further down the line of pathology than salsolinol or N-methyl-salsolinol. Instead, we will use salsolinol to induce PD in rats, which has been previously done in a number studies (8,12). If we are indeed able to inhibit SNMT in this manner, we will proceed to test the hypothesis that N-methyl-salsolinol causes PD by subjecting salsolinol treated rats to a standard battery of tests used to assess PD progression (13). Importantly, counts of surviving dopaminergic neurons in rats treated with SNMT inhibitors versus a control group will be proof of the efficacy of our compounds.

List of References

1. Surendran, S., & Rajasankar, S. (2010). Parkinson’s disease: oxidative stress and therapeutic approaches. Neurological Sciences, 31(5), 531-540.

2. Chun, H. S., Gibson, G. E., deGiorgio, L. A., Zhang, H. H., Kidd, V. J., & Son, J. H. (2001). Dopaminergic cell death induced by MPP[sup +], oxidant and specific neurotoxicants shares the common molecular mechanism. Journal Of Neurochemistry, 76(4), 1010-1021.

3. Akao Y, Maruyama W, Yi H, Shamoto-Nagai M, Youdim MB, Naoi M. (2002). An anti-Parkinson's disease drug, N-propargyl-1(R)-aminoindan (rasagiline), enhances expression of anti-apoptotic bcl-2 in human dopaminergic SH-SY5Y cells. Neurosci Lett., 326(2):105-8.

4. Mravec B. (2006). Salsolinol, a derivate of dopamine, is a possible modulator of catecholaminergic transmission: a review of recent developments. Physiol Res., 55(4):353-64.

5. Yi, H., Akao, Y., Maruyama, W., Chen, K., Shih, J., & Naoi, M. (2006). Type A monoamine oxidase is the target of an endogenous dopaminergic neurotoxin, N-methyl( R)salsolinol, leading to apoptosis in SH-SY5Y cells. Journal Of Neurochemistry, 96(2), 541-549.

6. Naoi M, Maruyama W, Nakao N, Ibi T, Sahashi K, Benedetti MS. (1998). (R)salsolinol N-methyltransferase activity increases in parkinsonian lymphocytes. Ann Neurol., 43(2):212-6.

7. Maruyama W, Strolin-Benedetti M, Naoi M. (2008). N-methyl(R)salsolinol and a neutral N-methyltransferase as pathogenic factors in Parkinson's disease. Neurobiology (Bp)., 8(1):55-68.

8. Copeland RL Jr, Leggett YA, Kanaan YM, Taylor RE, Tizabi Y. (2005). Neuroprotective effects of nicotine against salsolinol-induced cytotoxicity: implications for Parkinson's disease. Neurotox Res., 8(3-4):289-93.

9. Drinkwater N, Vu H, Lovell KM, Criscione KR, Collins BM, Prisinzano TE, Poulsen SA, McLeish MJ, Grunewald GL, Martin JL. (2010). Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors. Biochem J., 431(1):51-61.

10. Hajduk, P. J., & Greer, J. (2007). A decade of fragment-based drug design: strategic advances and lessons learned. Nature Reviews Drug Discovery, 6(3), 211-219.

11. Yongqian, Z., Lin, W., Xiaoling, M., Jinyan, D., Yong, Z., Hong, Q., & … Yulin, D. (2011). Assessment of salsolinol N-methyltransferase activity in rat peripheral lymphocytes by liquid chromatography-electrospray time-of-flight mass spectrometry. Analytical & Bioanalytical Chemistry, 399(10), 3541-3545.

12. Banach T, Zurowski D, Gil K, Krygowska-Wajs A, Marszałek A, Thor PJ. (2006). Peripheral mechanisms of intestinal dysmotility in rats with salsolinol induced experimental Parkinson's disease. J Physiol Pharmacol., 57(2):291-300.

13. Emborg ME. (2004). Evaluation of animal models of Parkinson's disease for neuroprotective strategies. J Neurosci Methods., 139(2):121-43.

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