CN112843067B

CN112843067B - Human LRRK2 protein small-molecule inhibitor and application thereof

Info

Publication number: CN112843067B
Application number: CN201911175850.4A
Authority: CN
Inventors: 刘焕香; 谈硕彦; 彭立增; 姚小军
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2023-01-06
Anticipated expiration: 2039-11-26
Also published as: CN112843067A

Abstract

The invention provides a small molecule inhibitor taking a human LRRK2 protein as a target spot and application thereof, belonging to the technical field of pharmacy. The invention takes a kinase domain of LRRK2 as a target spot, constructs a three-dimensional structure of the kinase domain of LRRK2 by a homologous modeling method, selects a type I inhibitor binding pocket, carries out molecular docking by utilizing a Glide program, virtually screens more than 160 ten thousand compounds to obtain a small molecular inhibitor LY2019-005 targeting human LRRK2 protein, carries out a kinase activity inhibition experiment, verifies the inhibition effect of the compound LY2019-005 on the LRRK2 kinase activity, carries out molecular dynamics simulation research, and clarifies the action mechanism of the compound LY2019-005 and the target protein on a molecular level. The discovered small molecules and the medicinal salts thereof can provide a basis for developing new drugs for treating LRRK2 related diseases, such as Parkinson's disease, alzheimer's disease, inflammatory bowel disease or leprosy. The lead compound can be further structurally optimized, and has a good application prospect.

Description

Human LRRK2 protein small-molecule inhibitor and application thereof

Technical Field

The invention belongs to the technical field of biochemical pharmacy, and particularly relates to a lead compound for inhibiting the activity of human LRRK2 kinase by taking leucine-rich repeat sequence kinase 2 (LRRK 2) protein as a target and application of a medicinal salt thereof in preparation of LRRK2 related disease drugs.

Background

LRRK2 is a large protein containing 2527 amino acids, belongs to the ROCO protein Kinase family, and comprises six domains of ARM, ANK, LRR, roc-COR, kinase, WD40 and the like. The Roc-COR domain has GTPase activity and binds to guanine nucleotides via the phosphate binding motif (P-loop) region and hydrolyzes GTP (Biosa A et al, human molecular genetics.2013,22 (6): 1140-56). The Kinase domain has ATP Kinase activity. Kinase domains have been shown to phosphorylate a variety of cellular proteins in vitro, but endogenous substrates have not yet been established. LRRK2 has been found to be widely expressed in brain, heart, kidney, lung, liver and some immune cells, and is widely involved in cellular processes including mitochondrial maintenance, synaptic vesicle cycling, autophagy, lysosomal biology, cytoskeletal regulation, neurite outgrowth regulation and translational control (Wallings R et al, the FEBS journal,2015,282 (15): 2806-26). The most common pathogenic mutation of LRRK2 is the G2019S mutation of the kinase domain, and the G2019S mutation in LRRK2 is reported to cause 5-6% familial and 1-2% sporadic parkinson' S disease, a risk factor for sporadic PD (Chen J et al, european neurology,2018,79 (5-6): 256-65.). Parkinson's Disease (PD) is a chronic progressive neurodegenerative disease, has a very complex pathogenesis and is the result of the combined action of multiple factors such as genes, environment and the like. Approximately 80% of PD patients are of unknown etiology and are idiopathic, while the remaining 20% of cases are considered hereditary (Bhat S et al Computers in biology and medicine,2018, 102. Of these, LRRK2 mutations are the most common cause of autosomal dominant inheritance of Parkinson's disease. LRRK2 mutations account for 1% in sporadic PD cases, 5% in all familial PD cases, and 2-3% of all PD cases (Alessi DR et al Science,2018,360 (6384): 36-37). The G2019S mutation increased the protein kinase activity of LRRK2 by about 2-fold, and it is presumed that the G2019S mutation caused the increase of the kinase activity of LRRK2 by enhancing the autophosphorylation level of LRRK2 and the phosphorylation level of a substrate, affecting synaptic transmission and autophagy functions, and inhibiting the growth of neurites, resulting in a defect in the growth of dopamine neurons (Steger M et al, eLife,2016,5.Cookson MR et al, nature reviews Neuroscience,2010,11 (12): 791-7). Recent studies have found that idiopathic PD patients without mutations also have LRRK2 protein overactivation, autophosphorylation, or increased expression, suggesting that LRRK2, whether mutated or not, plays a key role in PD development, participating in pathogenic processes (Di Maio R et al Science metabolic media, 2018,10 (451)). Taken together, elevated kinase activity of LRRK2 and its mutants is a significant cause of neuronal death, suggesting that controlling kinase activity of LRRK2 is a strategy for treating PD.

In addition, it has been suggested that LRRK2 may be involved in the activation and maturation of immune cells within the immune system, controlling free radical burst against pathogens in macrophages, and modulating neuroinflammation through cytokine signaling. The role of LRRK2 in immune response modulation may demonstrate The genetic relevance of LRRK2 to susceptibility to inflammatory bowel disease and leprosy (Wallings R et al. The FEBS journal.2015,282 (15): 2806-26.Hui KY et al. Sci Transl Med.2018Jan 10 (423). Furthermore, LRRK2 is able to phosphorylate the APP protein, the precursor of β amyloid protein causing alzheimer's disease, and the phosphorylated APP protein is toxic to cells after entering the nucleus (Chen ZC et al Sci signal.2017jul 18 (488)), suggesting a correlation of LRRK2 with alzheimer's disease.

The LRRK2 inhibitor can achieve the purpose of treating Parkinson's disease, alzheimer's disease, inflammatory bowel disease and leprosy by inhibiting the activity of LRRK2 kinase, most of LRRK2 inhibitors researched at present are ATP competitive type I kinase inhibitors, and most of LRRK2 inhibitors have the problems of insufficient specificity, incapability of passing through a blood brain barrier, certain toxicity and the like (Liu M et al, biochemistry.2013,52 (10): 1725-36), and an improved LRRK2 inhibitor with a novel framework still needs to be found.

Virtual screening (virtual screening) is a common method in the development of new drugs, and the method screens out chemical molecules with possible therapeutic effects by selecting a small molecule database and performing a calculation method, researches the binding effect of the chemical molecules and drug targets, and finally selects a compound with highest interaction binding force as a lead compound and uses the compound as a next step for research. Compared with the physical drug screening method, the virtual screening method has obvious advantages of using a computer, finding new candidate drugs more quickly and economically, reducing the cost of drug screening and shortening the research and development period of new drugs.

Disclosure of Invention

The invention aims to provide a lead compound targeting human LRRK2 protein and application of a medicinal salt thereof in preparing an LRRK2 kinase inhibitor. Wherein the LRRK2 mediated disease is selected from at least one of the following diseases: parkinson's disease, alzheimer's disease, inflammatory bowel disease and leprosy.

On the basis of research on a type I inhibitor binding pocket of a target LRRK2 protein, a small molecule inhibitor LY2019-005 of a target human LRRK2 protein with a new structural entity is obtained by screening, and the small molecule inhibitor of the target human LRRK2 protein is a compound of a small molecule structure aiming at a binding site of LRRK2 and ATP.

The invention utilizes a homologous modeling method to construct a three-dimensional structure of a human LRRK2 kinase domain, and finally obtains an active compound LY2019-005 targeting human LRRK2 with pharmacological activity through virtual screening based on the LRRK2 protein structure, and through multi-factor comprehensive analysis such as Ribes-based rule screening, scoring of scoring function, combination with free energy calculation, inhibition experiment on human LRRK2 protein kinase activity and the like.

Experiments prove that the compound LY2019-005 has a remarkable effect of inhibiting the activity of human LRRK2 kinase in vitro, and has strong inhibition on the activity of LRRK2 protein kinase of both wild type and mutant type (G2019S mutant type IC50=378nM, and wild type IC50=424 nM). The compound of the invention can be further structurally optimized to prepare a preparation or a medicament for treating LRRK2 protein related diseases (including Parkinson's disease, alzheimer's disease, inflammatory bowel disease and leprosy).

The compound targeting the human LRRK2 protein has the advantages that:

1. the compound has stronger effects of simultaneously inhibiting the activities of LRRK2 wild type and G2019S mutant kinases and targeting LRRK2 protein;

2. the compound LY2019-005 and the medicinal salt derivative thereof can be used for preparing a preparation or a medicament for treating diseases (including Parkinson disease, alzheimer disease, inflammatory bowel disease or leprosy) with LRRK2 protein as a target;

3. by a molecular dynamics simulation method, the interaction mode of the compound LY2019-005 and LRRK2 is researched, the reason of the activity of the compound is explained, and a foundation is provided for the structural modification of the medicine in the future.

For ease of understanding, the compounds of the invention targeting the human LRRK2 protein will be described in detail below with specific figures and examples. It is specifically noted that the specific examples and figures are for illustrative purposes only and it will be apparent to those skilled in the art that, in light of the description herein, various modifications and changes can be made in the invention which are within the scope of the invention.

Drawings

FIG. 1 (A) shows domain composition of LRRK2 protein and common pathogenic mutations;

FIG. 1 (B) is a homologously modeled LRRK2 kinase structure in which small molecules shown in stick form are small molecule inhibitors of the template self-assembly;

FIG. 2 is the chemical structural formula of compound LY2019-005 of the present invention.

FIG. 3 shows the inhibition of kinase activity by small molecule compound LY2019-005 at the protein level. The left panel shows the inhibition curve of small molecule compounds on LRRK2 wild-type protein. The right graph shows the inhibition curve of the small molecule compound on the LRRK2 mutant G2019S protein.

FIG. 4 shows a representative conformation of the binding pattern of the LRRK2 kinase binding pocket to the small molecule compound LY2019-005. The upper graph shows the binding mode of the small molecule compound and the G2019S mutant; the lower panel shows the hydrogen bonding interaction of small molecule compounds with wild type, in which residue 72 of the homology modeling protein (residue 1950 corresponding to the full length LRRK2 protein) forms with small molecule compounds.

Detailed Description

Example 1

1. High-throughput virtual screening method of small molecule compound LY2019-005

(1) Protein preparation

Since the structure of the research target is unknown, a homologous modeling method is needed to build a kinase domain model of LRRK 2. According to the existing mixed-lineage kinase MLK1 (mixed-linkage kinases, MLK 1) crystal structure (PDB code:3 dtc) as a template (identities 31%; aggressive 51%), use was made of

The Prime module of the software constructs the LRRK2 kinase domain (amino acid sequence 1879-2138). In order to obtain a more stable and reasonable LRRK2 kinase domain structure, the molecular dynamics simulation of 200 ns is further carried out on the structure after homologous modeling. Then, stable 3 representative protein structures are obtained through cluster analysis, a re-docking method and the investigation of a Laplace diagram and are used for virtual screening in the future.

(2) Ligand preparation

Three-dimensional structures of approximately 1,600,000 compounds were downloaded from SPECS and chemdiv chemical databases

The Lig Prep module in Maestro software is further structurally processed and optimized to minimize various ionic states, tautomerism, stereo configurations, etc. of the structure, resulting in a ligand with optimal structure and lowest energy.

(3) Molecular docking-based virtual screening

By using

And carrying out molecular docking on the Glide module of the software. Binding pockets are defined based on the type i inhibitor binding sites, and default parameters are used in the generation of the grid points. The accuracy of the progressive screening was set to 10% before HTVS retention, 10% before SP retention, and 50% before XP retention. By using

The Canvas module in the software clustered the resulting compounds and selected 28 compounds (see structural formula below) for the next activity assay, 26 of which were from the Chemdiv database and 2 from the SPECS library.

2. Inhibitory effect of virtually screened 28 small molecule compounds on LRRK2 kinase activity

The ability of 28 small-molecule compounds to inhibit the activity of LRRK2 kinase is detected by using a Caliper mobility shift assay method, and the fact that the small-molecule compounds LY2019-005 have good kinase activity inhibition effects on LRRK2 proteins of wild type G2019S and mutant G2019S is found (as shown in figure 3), and half effective inhibition concentrations of the small-molecule compounds LY2019-005 in inhibiting LRRK2 kinase are 424nM (wild type) and 378nM (G2019S mutant).

3. Molecular dynamics simulation study

Small molecule compound LY2019-005 is screened out based on structure virtualization, and enzyme activity evaluation experiments prove that LRRK2 kinase activity can be effectively inhibited. An Amber14 molecular dynamics simulation method is used for researching an interaction mechanism of a small molecular compound LY2019-005 with WT and G2019S LRRK2, and the fact that a residue 72 of an LRRK2 kinase domain (corresponding to a residue 1950 of a full-length LRRK2 protein) and the compound LY2019-005 can form stable hydrogen bond interaction is found, so that the reason for good kinase inhibition activity of the compound LY2019-005 is explained from a theoretical point of view, and guidance is provided for further modification of the small molecular compound.

Claims

1. The application of a compound LY2019-005 and a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting the activity of human LRRK2 kinase, wherein the medicament is used for treating Parkinson's disease, and the compound LY2019-005 has the following structural formula: