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Cancer is a multifactorial disease that involves numerous genetic defects and characterized by abnormal growth of cells. It may affect almost all types of tissues in human beings and over 200 types of cancer has been identified, characterized and reported. Cancer is a leading cause of death worldwide. From a total of 58 million deaths worldwide in 2005, cancer accounts for 7.6 million (or 13%) of all deaths. The main types of cancer leading to overall cancer mortality are:
Lung (1.3 million deaths/year);
Stomach (almost 1 million deaths/year);
Liver (662,000 deaths/year);
Colon (655,000 deaths/year) and
Breast (502,000 deaths/year).
More than 70% of all cancer deaths in 2005 occurred in low and middle income countries. Deaths from cancer in the world are projected to continue rising, with an estimated 9 million people dying from cancer in 2015 and 11.4 million dying in 20301.
Major risk factors of cancer includes Diet, tobacco and smoking, chronic infection and inflammation, and hormonal imbalance. Less important risk factors includes occupation, pollution, exposure to sunlight and hereditary factors. These factors interferes with the transcription of genes controlling cell cycle events . An imbalance between activator and repressor gene controlling cell cycle events is the major reason leading to cancer. Such imbalance happens either due to the interaction of external factor (i) with the signal transduction mechanism in cytoplasm which in turn affects nuclear events or (ii) with DNA in nucleus directly, which results in mutation of genes leading to cancer2. The mutation may lead to any one or more of the following events in the pathogenesis of cancer,
Activation of ONCOGENES,
Repression of ANTI-ONCOGENES (Tumor Suppressor Genes),
Suppression of APOPTOSIS,
Suppression of DNA repair mechanism and
In order to suppress the progression of cancer, an anticancer agent(s) must be designed to address more than one of the above listed factors. Till late 1970, medicinal chemists designed their drugs for an ailment based on the earlier literature available and tested their molecules in animal model. Success of such a molecule is purely based on their effect due to its action on multiple targets which were not known at that time. Most of the drugs were good for the treatment but at the same time with some major side effects. After 1980, as field of biochemistry and biotechnology started revealing the molecular mechanism underlying a disease, people began to explore it to understand mechanism of action of a drug molecule with reason for its unwanted side effects at molecular level. This is the time people started calling the less specific drugs as "DIRTY or PROMISCUOUS" drugs and looking forward to design a molecule to target a specific protein responsible for a disease pathogenesis. The concept of "MAGIC BULLET" to hit a specific target started dominating drug discovery arena from then to till date3.
But 20 years down the line people started realizing the fact that this target based approach does not always guarantee success in case of multifactorial disease such as cancer, neurodegenerative disorders, cardiovascular disorders, HIV etc.,. For the treatment of such a disease one need to depend on more than one "Magic Bullet" at all the time which complicates the things at all the level.
At industry level, investment for drug discovery process multiplies depending on the number of targets they are selecting for a particular disease,
At clinical level, more drugs means more side effects with increased chance of drug interaction, and
At consumer level, problem of affordability and compliance.
This fact made the scientists in the industry as well as in academic to think of designing a drug to hit multiple target responsible for a disease4. With the knowledge of complex interdependent factors responsible for pathogenesis of disease and computational chemistry, scientists have already began to design a molecule targeting more than one protein. Simple concept is to include all the pharmacophoric characters of inhibitors of targets selected in a single molecule. It is evident from the fact that more than 300 articles has been published on multifunctional molecules between the period 1990-2004. Compared with articles reported for magic bullets, the number of articles reported for multifunctional molecules were very less during this period but increasing in percentage every year. In next few years the drug discovery arena will witness a sea change with reversal of the pattern which is existing today5.
Our research proposal is to design and synthesize bifunctional anticancer molecule targeting both Histone deacetylase and Ribonucleotide reductase.
The basic protein Histone is an octomer, which is rich in histidine and lysine. Histone plays a major role in the organization of DNA into nucleosome-chromatin-chromosome. This organization restricts the binding of transcriptional activator/repressor to DNA. In order to favor the binding of transcriptional activator/repressor to DNA, chromatin has to undergo remodeling(chromatin dynamics). Post-translational modification of Histone is one of the way it adopts for the purpose. The site specific post translational modification of histone tail in a specific pattern permits the binding or unbinding of specific transcriptional activator/repressor leading to initiate/repress the transcription of a specific gene is known as HISTONE CODE. The well characterized Post-translational modification is Acetylation/Deacetylation of Histone tail (N-terminus-lysine residues). Balanced activity of Histone acetyl transferase (HAT) and Histone deacetylase (HDAC) controls the transcription of genes responsible for normal cell cycle events. Over expression of HDAC represses the transcription of tumor suppressor gene which regulates the entry of cell from G1 phase to S phase leading uncontrolled cell division and results in cancer. Over expression of HDAC has been reported in most of the cancer types. Inhibition of this enzyme has been shown to suppress cancer in in vitro as well as in vivo models. HDAC inhibitors arrests cell cycle at G1 to S and induces Differentiation and Apoptosis. Almost all HDAC inhibitors activate transcription of the Cyclin-dependent kinase (CDK) inhibitor WAF1 (also known as CIP1, p21; encoded by the CDKN1A locus), which can inhibit Cyclin E-CDK2 and Cyclin A-CDK2, and many HDAC inhibitors down regulate Cyclin A and D, which prevents entry of cell from G1 phase to S phase.
Ribonucleotide reductase (RR) catalyzes the reduction of Ribonucleotide to their corresponding deoxyribonucleotides, which are the building blocks for DNA replication and repair in all living cells. Since the reduction of Ribonucleotide is the rate-limiting step of DNA synthesis, inactivation of RR stops DNA synthesis, which inhibits cell proliferation. The important role of RR in DNA synthesis and repair has made it an important target for anticancer. Increased RR activity has been associated with malignant transformation and cancer metastasis. The effect of RR inactivation in cells includes decreases of intracellular concentrations of the Deoxy nucleotide triphosphates (dNTPs), inhibition of DNA synthesis, inhibition of DNA repair in quiescent cells, and cell cycle arrest at S-phase and apoptosis.
Design of hybrid inhibitor:
Hydroxamates are the major Class of agents reported as HDAC inhibitors. Hydroxamate Pharmacophore has three basic components (a) a hydroxamic acid moiety; required for metal ion chelation (Zn2+), (b) a hydrophobic spacer; required to interact with the hydrophobic channel and (c) a hydrophobic cap; which determines selectivity8. Many analogues were reported with the various changes in the last two components, (b) Spacer and (c) Cap. Many linear and cyclic hydrophobic spacers (alicyclic and aromatic) were reported with many different heteroaromatic rings and macro cyclic rings as hydrophobic cap9. Small molecule inhibitor of RR can be tried in the place of cap of HDAC inhibitors. Design and synthesis of a Hybrid molecule with both pharmacophoric characteristics of HDAC inhibitors and RR inhibitors are expected to be more potent and effective.
To design Hybrid Molecule targeting both HDAC and CDK from the molecules reported earlier in the literature using QSAR/Docking Softwares, and to propose Novel Hybrid inhibitor.
To develop suitable synthetic methodology for the synthesis of proposed hybrid inhibitor.
To synthesize and characterize the proposed hybrid inhibitors.
To subject the synthesized hybrid inhibitors for in vitro enzyme based assays and then to cancer cell line based assays
To undertake further QSAR studies on Hybrid inhibitors.
III. Plan of work and Methodology:
Proposed activity will be carried out in three Phases.
Creating a Database of HDAC inhibitors and RR inhibitors, which are reported already with their IC50 value.
Carrying-out QSAR studies with the inhibitors reported already to develop a model using Open source QSAR Softwares.
Testing of designed hybrid inhibitors in the QSAR model developed and necessary manipulation will be done to get molecules with best predicted activity.
Further refinement will be carried by docking the molecules with their respective target protein using Open source Docking Softwares.
Novel Hybrid inhibitor will be proposed for synthesis.
Synthetic methodology will be developed and optimized for the synthesis of proposed Hybrid inhibitors.
Efforts will be made to synthesize and characterize (preliminary, TLC, MP, IR and CHNS analysis) all possible Hybrid inhibitors proposed.
Further characterization and screening of synthesized hybrid inhibitors will be carried out.
Characterization includes H1NMR, C13NMR and MS analysis and their interpretation.
Screening includes in vitro enzyme based assay followed by cancer cell line assay.
Efforts will be made to study and develop a QSAR model for the newly synthesized Novel hybrid inhibitors using Open source QSAR Softwares.
IV. Scope of proposed work:
The rationale approach to design a drug to hit multiple targets of cancer is the area yet to be explored. Any positive outcome of the proposed work will send a strong message to scientific community to approach drug design from a different direction which is exactly opposing the present form of drug discovery approach. Also this approach may reduce national economic burden due to any multifactorial disease such as cancer.
Hydroxamates Pharmacophore of HDAC inhibitors offers a greater opportunity to manipulate its Cap region without much disturbing its ability to inhibit HDAC. Other anticancer protein inhibitors can also be tried in the cap region of HDAC inhibitors.
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