COMPUTATIONAL AIDED DRUG DESIGN FOR CHRONIC MYELOID LEUKEMIA (CML)DISEASE

INTRODUCTION:-

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder characterized by the Philadelphia (Ph) chromosome, that results from a structural chromosomal instability caused by the translocation of a portion of chromosome 22 onto chromosome 9 (t[9,22]). This translocation leads to the fusion of the Breakpoint Cluster Region (BCR) gene to the Abelson Tyrosine Kinase (ABL1) proto-oncogene and the formation of the BCR-ABL1 oncogene.

Common Symptoms in patients with CML:-

Chronic myeloid leukaemia (CML) symptoms are frequently ambiguous and are frequently caused by other factors. They are as follows:

· Weakness,

· Fatigue,

· Sweating at night,

· Loss of weight,

· Fever,

· Bone ache (caused by leukaemia cells spreading from the marrow cavity to the surface of the bone or into the joint),

· A spleen enlargement (felt as a mass under the left side of the ribcage),

· Pain or a feeling of fullness in the stomach

Age Factor: Chronic myeloid leukaemia (CML) is a malignancy that affects white blood cells and progresses slowly over time. It may happen at any age, although it is more prevalent in older individuals between the ages of 60 and 65.

MOLECULAR PATHOPHYSIOLOGY OF DISEASE:-

BCR gene is an oligomerization domain and it has tyr177, serine, threonine kinase domains.
ABL gene contains SH3, SH2 domains and SH1 tyrosine kinase domain. Among all these domains SH3 domain is one of the important domains of the ABL gene since it is a negative regulator of the tyrosine kinase domain of ABL i.e. SH1. So, this SH3 will turns off the kinase activity of SH1 as an inhibitory action.

But after the translocation of the BCR-ABL fusion gene where the N-terminal part is of the BCR gene and the C-terminal part is for ABL-gene.

Here the oligomerization domain and tyr177 of the BCR gene interfere with the SH3 domain of the ABL gene and renders it non-functional and the SH3 domain no longer works which means it will not inhibit tyrosine kinase.

So without any regulation, the tyrosine kinase SH1 gets permanently on and activates the MAPK pathway, JAK-STAT pathway, and PI3 K pathway, leading to cell proliferation, survival, and metastasis.

KEGG Pathway of CML:-

TARGET IDENTIFICATION FOR DRUG DISCOVERY:-

The dual — targets have been chosen for the CML drug discovery.
The ABL1 (Abelson leukemia virus) and JAK2 (Janus Kinase 2) have been chosen as the targets based on the molecular pathway they are involved in and results in CML.

Reason for choosing ABL1 as target: As CML is caused due to the fusion of the BCR-ABL gene, targeting either BCR or ABL can stop the BCR-ABL enzyme from working which causes the CML cells to die.
As BCR and ABL are required for CML's pathogenesis by activating multiple intracellular signal transduction pathways such as Ras/Raf/MAPK and PI3K/AKT via its protein tyrosine kinase (PTK) interfering with the development of hematopoietic stem cells or progenitor cells. Thus, targeting the signaling pathways activated by BCR/ABL is a promising approach for drug development to treat CML.

Reason for choosing JAK2 as another target: JAK2 interacts directly with the C-terminal region of BCR-ABL and is a key interaction partner of BCR-ABL in CML. This complex enhances BCR-ABL TK activity and disrupts BCR-ABL-mediated signaling in BCR-ABL+ cells, possibly through direct phosphorylation of tyrosine 177 of BCR-ABL by JAK2.
In normal cells, nuclear translocation of STATs occurs exclusively after cytokine binding to receptors and is mediated by activation of the receptor-associated JAK kinases. By contrast, in CML, STATs seem to be activated in a JAK-independent manner through a direct association of STAT SH2 domains with phosphorylated tyrosine’s on BCR-ABL. Activation of STAT5 is at least partially responsible for protection from programmed cell death.
Although the important roles of JAK2 and STAT5 in LSC growth and survival and thus disease evolution are well appreciated, it remains unclear whether targeting of either JAK2 or STAT5 is meaningful and can be achieved using currently available drugs. Concerning STAT5, the major problem is that no specific and potent inhibitors have been developed far enough to reach the clinical application. In addition, STAT5 inhibition in LSCs alone may not be sufficient as other STAT molecules, such as STAT3, may compensate for a loss or suppression of STAT5. Because STAT3 is also downstream of JAK2 and has also been implicated in the pathogenesis of CML, targeting JAK2 may be a more logical approach as it suppresses the activity of both STAT3 and STAT5 in CML (stem) cells.

LIGAND DATA FOR BOTH THE TARGETS:

For ABL1 total of 3791 ligands based on their IC50 values have been downloaded from the BindingDB and ChEMBL.

For JAK2 total of 13807 ligands based on their IC50 values have been downloaded from the BindingDB and ChEMBL.

LEAD-MOLECULE GENERATION IN BOLTCHEM:

· PROPERTY PREDICTION AND VALIDATION OF PROPERTY:

The property prediction has been done for both targets based on their IC50 values. Their property has been validated between predicted once and novel molecule property.

JAK2 and ABL1 have been tested on their IC50 values in a natural log with minimum discrepancies in the input and output values, 1–2 points, which is the accepted amount for ongoing studies.

· SUBSTRUCTURE GENERATION:

The screening technique was developed to minimize the number of atom-by-atom matching. The aim is to pre-define a collection of substructures (chemical functional groups) as a screen to filter out unfeasible structural search options. The search engine avoids atom-by-atom matching of target structures that do not include all of the substructures found in the query structure.

The two targets of validated property data points have been submitted for substructure generation. As the JAK2 initial points were more than 13000 so the data points have been filtered out to 1000 based on their novelty and ABL1 has been submitted with 3000 data points. After submission of data, the substructures have been generated for both the targets

For ABL1 2330 substructures have been generated and for JAK2 984 substructures have been generated.

After merging the substructures in the required combination, BOLTCHEM can be used to generate lead molecules from the substructures found in a span of under 3–4 days, whereas manually it would take months to generate all possible lead molecules. We expect to get multiple lead molecules that can be used as bases for the drug for CML. Once the molecule similarity search has been run and the unique molecules have been identified, docking and molecular dynamics simulations can be run. The successful molecules will then be tested against existing drugs for similarity and effectiveness.

This way, by using the small molecule drug discovery platform — BOLTCHEM, the selected molecules will be ready for in vitro and in vivo studies in 2 months instead of the years it usually takes drug discovery to reach the testing phase.