Insight into the structural mechanism for PKBα allosteric inhibition by molecular dynamics simulations and free energy calculations

Protein kinase B (PKB/Akt) is an attractive target for the treatment of tumor. Unlike PKB's ATP-competitive inhibitors, its allosteric inhibitors can maintain PKB's inactive state via its binding in a pocket between PH domain and kinase domain, which specifically inhibit PKB by preventing the phosphorylations of Thr308 and Ser473. In the present studies, MD simulations were performed on three allosteric inhibitors with different inhibitory potencies (IC₅₀) to investigate the interaction modes between the inhibitors and PKBα. MM/GB(PB)SA were further applied to calculate the binding free energies of these inhibitors binding to PKBα. The computed binding free energies were consistent with the ranking of their experimental bioactivities. The key residues of PKBα interacting with the allosteric inhibitor were further discussed by analyzing the different interaction modes of these three inhibitors binding to PKBα and by calculating binding free energy contributions of corresponding residues around the binding pocket. The structural requirements were then summarized for the allosteric inhibitor binding to PKBα. A possible structural mechanism of PKBα inhibition induced by the binding of allosteric inhibitor was formulated. The current studies indicate that there should be an optimum balance between the van der Waals and total electrostatic interactions for further designing of PKBα allosteric inhibitors.     

A structural insight into the inhibitory mechanism of an orally active PI3K/mTOR dual inhibitor,PKI-179 using computational approaches

The PI3K/AKT/mTOR signaling pathway has been identified as an important target for cancer therapy. Attempts are increasingly made to design the inhibitors against the key proteins of this pathway for anti-cancer therapy. The PI3K/mTOR dual inhibitors have proved more effective than the inhibitors against only single protein targets. Recently discovered PKI-179, an orally effective compound, is one such dual inhibitor targeting both PI3K and mTOR. This anti-cancer compound is efficacious both in vitro and in vivo. However, the binding mechanisms and the molecular interactions of PKI-179 with PI3K and mTOR are not yet available. The current study investigated the exact binding mode and the molecular interactions of PKI-179 with PI3Kγ and mTOR using molecular docking and (un)binding simulation analyses. The study identified PKI-179 interacting residues of both the proteins and their importance in binding was ranked by the loss in accessible surface area, number of molecular interactions of the residue, and consistent appearance of the residue in (un)binding simulation analysis. The key residues involved in binding of PKI-179 were Ala-805 in PI3Kγ and Ile-2163 in mTOR as they have lost maximum accessible surface area due to binding. In addition, the residues which played a role in binding of the drug but were away from the catalytic site were also identified using (un)binding simulation analyses. Finally, comparison of the interacting residues in the respective catalytic sites was done for the difference in the binding of the drug to the two proteins. Thus, the pairs of the residues falling at the similar location with respect to the docked drug were identified. The striking similarity in the interacting residues of the catalytic site explains the concomitant inhibition of both proteins by a number of inhibitors. In conclusion, the docking and (un)binding simulation analyses of dual inhibitor PKI-179 with PI3K and mTOR will provide a suitable multi-target model for studying drug–protein interactions and thus help in designing the novel drugs with higher potency.     

Molecular docking/dynamics studies of Aurora A kinase inhibitors

The binding modes of a known 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole, quinazoline, pyrimidine and indolinone series of Aurora A kinase inhibitors have been studied using molecular docking and molecular dynamics (MD) simulations. Crystallographic bound compound 8 was precisely predicted by our docking procedure as evident from 0.43 Å root mean square (rms) deviations. In addition compound 25 (AZ_68) has been successfully cross-docked within the Aurora A kinase active site, which was pre-organized for inhibitor 8. We found four key sites (A: solvent-exposed front pocket, B: hinge region, C: selectivity pocket and D: solvent-exposed phosphate binding region) of the Aurora A kinase contributing towards the binding of these compounds. We suggest that the small hydrophobic substituents at C-6 position of pyrrolopyrazole nucleus (in compounds 1–8); C-6 and C-7 positions of the quinazoline moiety (in compounds 9–23); C-2 position of the quinazoline and C-4 position of the pyrimidine (in compound 25) could be more effective and selective through increased hydrophobic contacts and selectivity pocket interactions with these modifications of Aurora A kinase inhibitors. Five representative complexes were subjected to 1000 ps of MD simulation to determine the stability of the predicted binding conformations. The low value of the root mean square deviations (ranging from 0.725 to 1.820 Å) between the starting complex structure and the energy minimized final average complex structure suggests that the Glide Extra Precision (XP) derived docked complexes are in a state of near equilibrium. The structure-based drug design strategy described in this study will be highly useful for the development of new inhibitors with high potency and selectivity.     

Structure-based drug design to the discovery of new 2-aminothiazole CDK2 inhibitors

N-(5-Bromo-1,3-thiazol-2-yl)butanamide (compound 1) was found active (IC50=808 nM) in a high throughput screening (HTS) for CDK2 inhibitors. By exploiting crystal structures of several complexes between CDK2 and inhibitors and applying structure-based drug design (SBDD), we rapidly discovered a very potent and selective CDK2 inhibitor 4-[(5-isopropyl-1,3-thiazol-2-yl)amino] benzenesulfonamide (compound 4, IC50=20 nM). The syntheses, structure-based analog design, kinases inhibition data and X-ray crystallographic structures of CDK2/inhibitor complexes are reported.     

Structural model of the Plasmodium CDK, Pfmrk, a novel target for malaria therapeutics

Malaria, with 300-500 million clinical cases resulting in 1-3 million fatalities a year, is one of the most deadly tropical diseases. As current antimalarial therapeutics become increasingly ineffective due to parasitic resistance, there exists an urgent need to develop and pursue new therapeutic strategies. Recent genome sequencing and molecular cloning projects have identified several enzymes from Plasmodium (P.) falciparum that may represent novel drug targets, including a family of proteins that are homologous to the mammalian cyclin-dependent kinases (CDKs). CDKs are essential for the control of the mammalian cell cycle and, based on the conservation of the CDKs across species, the plasmodial CDKs are expected to play a crucial role in parasitic growth. Here we present a 3D structural model of Pfmrk, a putative human CDK activating kinase (CAK) homolog in P. falciparum. Notable features of the present structural model include: (1) parameterization of the Mg2+ hexacoordination system using ab initio quantum chemical calculations to accurately represent the ATP-kinase interaction; and (2) comparison between the docking scores and measured binding affinities for a series of oxindole-based Pfmrk inhibitors of known activity. Detailed analysis of inhibitor-Pfmrk binding interactions enabled us to identify specific residues (viz. Met66, Met75, Met91, Met94 and Phe143) within the Pfmrk binding pocket that may play an important role in inhibitor binding affinity and selectivity. The availability of this Pfmrk structural model, together with insights gained from analysis of ligand-receptor interactions, should promote the rational design of potent and selective Pfmrk inhibitors as antimalarial therapeutics.     

Exploring putative inhibitors of Death Associated Protein Kinase 1 (DAPK1) via targeting Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN) substrate recognition motifs

Recently, a new signaling complex Death Associated Protein Kinase 1 (DAPK1) ̶ N-methyl-D-aspartate receptor subtype 2B (NMDAR2B or NR2B) engaged in the neuronal death cascade was identified and it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via over-activation of NMDA receptors. An acute brain injury, such as stroke, is a serious life-threatening medical condition which occurs due to poor blood supply to the brain and further leads to neuronal cell death. During a stroke, activated DAPK1 migrates towards the extra-synaptic site and binds to NR2B subunit of NMDA receptor. It is this DAPK1-NR2B interaction that arbitrates the pathological processes like apoptosis, necrosis, and autophagy of neuronal cells observed in stroke injury, hence we aimed to inhibit this vital interaction to prevent neuronal damage. In the present study, using PubChem database, we applied an integrative approach of virtual screening and molecular dynamic simulations and identified a potential lead compound 11 that interrupts DAPK1-NR2B interaction by competing with both ATP and substrate for their binding sites on DAPK1. This inhibitor was found potent and considerably selective to DAPK1 as it made direct contact with the ATP binding sites as well as substrate recognition motifs: Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN). Further in vitro and in vivo experiments are demanded to validate the efficacy of compound 11 nevertheless, it can be considered as suitable starting point for designing DAPK1 inhibitors.     

2,4-Diamino-5-(2'-arylpropargyl)pyrimidine derivatives as new nonclassical antifolates for human dihydrofolate reductase inhibition

Dihydrofolate reductase (DHFR) has been a well-recognized target for the development of therapeutics for human cancers for several decades. Classical inhibitors of DHFR use an active transport mechanism to gain access to the cell; disabling this mechanism creates a pathway for resistance. In response, recent research focuses on nonclassical lipid-soluble DHFR inhibitors that are designed to passively diffuse through the membrane. Here, a new series of propargyl-linked antifolates are investigated as potential nonclassical human DHFR inhibitors. Several of these compounds exhibit potent enzyme inhibition with 50% inhibition concentration values under 500 nM. Molecular docking investigations show that the compounds maintain conserved hydrogen bonds between the pyrimidine ring and the enzyme as well as form van der Waals interactions with critical residues in the active site. Interestingly, the most potent compound, 2,4-diamino-5-(3-(3,4,5-trimethoxyphenyl)prop-1-ynyl)-6-ethylpyrimidine (compound 35), is 3500-fold more potent than trimethoprim, a potent inhibitor of bacterial DHFR but weak inhibitor of human DHFR. The two structural differences between compound 35 and trimethoprim show that the propargyl linkage and the substitution at C6 of the pyrimidine ring are critical to the formation of contacts with Thr 56, Ser 59, Ile 60, Leu 22, Phe 31 and Phe 34 and hence, to enhancing potency. The propargyl-linked antifolates are efficient ligands with a high ratio of potency to the number of non-hydrogen atoms and represent a potentially fruitful avenue for future development of antineoplastic agents.     

In silico design: Extended molecular dynamic simulations of a new series of dually acting inhibitors against EGFR and HER2

Based on the hit structures that have been identified in our previous studies against EGFR and HER2, new potential inhibitors that share the same scaffold of the hit structures are designed and screened in silico. Insights into understanding the potential inhibitory effect of the new inhibitors against both EGFR and HER2 receptors is obtained using extended molecular dynamics (MD) simulations and different scoring techniques. The binding mechanisms and dynamics are detailed with respect to two approved inhibitors against EGFR (lapatinib) and HER2 (SYR127063). The best scoring inhibitor (T9) is chosen for additional in silico investigation against both the wild-type and T790M mutant strain of EGFR and the wild-type HER2. The results reveal that certain substitution patterns increase the stability and assure stronger binding and higher H-bond occupancy of the conserved water molecule that is commonly observed with kinase crystal structures. Furthermore, the new inhibitor (T9) forms stable interactions with the mutant strain as a direct consequence of the enhanced ability to form additional hydrogen bonding interactions with binding site residues.     

Conserved water mediated H-bonding dynamics of Ser117 and Thr119 residues in human transthyretin–thyroxin complexation: Inhibitor modeling study through docking and molecular dynamics simulation

Transthyretin (TTR) is a protein whose aggregation and deposition causes amyloid diseases in human beings. Amyloid fibril formation is prevented by binding of thyroxin (T4) or its analogs to TTR. The MD simulation study of several solvated X-ray structures of apo and holo TTR has indicated the role of a conserved water molecule and its interaction with T4 binding residues Ser117 and Thr119. Geometrical and electronic consequences of those interactions have been exploited to design a series of thyroxin analogs (Mod1–4) by modifying 5′ or 3′ or both the iodine atoms of thyroxin. Binding energy of the designed ligands has been calculated by docking the molecules in tetrameric structure of the protein. Theoretically investigated pharmacological parameters along with the binding energy data indicate the potentiality of 3′,5′-diacetyl-3,5-dichloro-l-thyronine (Mod4) to act as a better inhibitor for TTR-related amyloid diseases.     

Exploring the selectivity of inhibitor complexes with Bcl-2 and Bcl-XL: A molecular dynamics simulation approach

B-cell lymphoma 2 (Bcl-2) family proteins are potential drug targets in cancer and have a relatively flat and flexible binding site. ABT-199 is one of the most promising selective Bcl-2 inhibitors, and A-1155463 selectively inhibits Bcl-XL. Although the amino acid sequences of the binding sites of these two inhibitors are similar, the inhibitors selectively bind the target protein. In order to determine the origin of the selectivity of these inhibitors, we conducted molecular dynamics simulations using protein-inhibitor modeling. We confirmed that ASP103 of Bcl-2 is a key residue and that hydrogen bonding between ASP103 and ABT-199 confers the Bcl-2 selectivity of this inhibitor. For Bcl-XL selectivity, the secondary structure of α-helix 3 is a key factor. PHE105, SER106, and LEU108 in the loose α-helix 3 interact with A-1155463 to confer Bcl-XL selectivity. These findings provide important insights into the molecular mechanisms of selective inhibitors of Bcl-2 family proteins.     

Structure-guided cancer blockade between bioactive bursehernin and proteins: Molecular docking and molecular dynamics study

Bursehernin (5′-desmethoxyyatein) is a natural lignan, which has anti-tumor activity in vitro. In this study, the binding-inhibitory effects of bursehernin were screening on selected 80 proteins associated with cancer pathway. The computational analysis suggested inhibitory effect due to bursehernin towards proteins related to cancer proliferation, including FMS kinase receptor, heat shock protein 90-α (Hsp90-α), adenylate cyclase 10 (ADCY10), mitogen-activated protein kinase kinase (MEK1), and α-tubulin. Moreover, bursehernin could interfere with cell cycle progression via binding to cyclin B proteins. Among all screened proteins, the compound showed an interesting binding affinity to the FMS kinase receptor. The binding mode studies by molecular dynamic technique showed that aromatic ring of bursehernin compound was responsible for compound-protein interaction through pi-pi stacking with Tyr105 and Phe178 of the FMS kinase receptor. This study suggests that bursehernin has potential for development as an anti-tumor agent with an anti-proliferation, and cell cycle arrest inducing, although further studies are needed.     

Can structural features of kinase receptors provide clues on selectivity and inhibition? A molecular modeling study

Cancer is a complex disease resulting from the uncontrolled proliferation of cell signaling events. Protein kinases have been identified as central molecules that participate overwhelmingly in oncogenic events, thus becoming key targets for anticancer drugs. A majority of studies converged on the idea that ligand-binding pockets of kinases retain clues to the inhibiting abilities and cross-reacting tendencies of inhibitor drugs. Even though these ideas are critical for drug discovery, validating them using experiments is not only difficult, but also in some cases infeasible. To overcome these limitations and to test these ideas at the molecular level, we present here the results of receptor-focused in-silico docking of nine marketed drugs to 19 different wild-type and mutated kinases chosen from a wide range of families. This investigation highlights the need for using relevant models to explain the correct inhibition trends and the results are used to make predictions that might be able to influence future experiments. Our simulation studies are able to correctly predict the primary targets for each drug studied in majority of cases and our results agree with the existing findings. Our study shows that the conformations a given receptor acquires during kinase activation, and their micro-environment, defines the ligand partners. Type II drugs display high compatibility and selectivity for DFG-out kinase conformations. On the other hand Type I drugs are less selective and show binding preferences for both the open and closed forms of selected kinases. Using this receptor-focused approach, it is possible to capture the observed fold change in binding affinities between the wild-type and disease-centric mutations in ABL kinase for Imatinib and the second-generation ABL drugs. The effects of mutation are also investigated for two other systems, EGFR and B-Raf. Finally, by including pathway information in the design it is possible to model kinase inhibitors with potentially fewer side-effects.     

Identification of adenine nucleotide translocase 4 inhibitors by molecular docking

The protein adenine nucleotide translocase (ANT) is localized in the mitochondrial inner membrane and plays an essential role in transporting ADP into the mitochondrial matrix and ATP out from the matrix for cell utilization. In mammals there are four paralogous ANT genes, of which ANT4 is exclusively expressed in meiotic germ cells. Since ANT4 has been shown essential for spermatogenesis and male fertility in mice, inhibition of ANT4 appears to be a reasonable target for male contraceptive development. Further, in contrast to ANT1, ANT2 and ANT3 that are highly homologous to each other, ANT4 has a distinguishable amino acid sequence, which serves as a basis to develop a selective ANT4 inhibitor. In this study, we aimed to identify candidate compounds that can selectively inhibit ANT4 activity over the other ANTs. We used a structure-based method in which ANT4 was modeled then utilized as the basis for selection of compounds that interact with sites unique to ANT4. A large chemical library (>100,000 small molecules) was screened by molecular docking and effects of these compounds on ADP/ATP exchange through ANT4 were examined using yeast mitochondria expressing human ANT4. Through this, we identified one particular candidate compound, [2,2′-methanediylbis(4-nitrophenol)], which inhibits ANT4 activity with a lower IC₅₀ than the other ANTs (5.8 μM, 4.1 μM, 5.1 μM and 1.4 μM for ANT1, 2, 3 and 4, respectively). This newly identified active lead compound and its chemical structure are expected to provide new opportunities to optimize selective ANT4 inhibitors for contraceptive purposes.     

Characteristics of the Plasmodium falciparum PK5 ATP-binding site: implications for the design of novel antimalarial agents

Increasing worldwide resistance of Plasmodium falciparum (P. falciparum) to traditional chemotherapy strategies such as chloroquine and mefloquine demonstrates the urgent need for the discovery of novel chemotherapeutic agents in the fight against malaria. The recent discovery of P. falciparum Protein Kinase 5 (PfPK5) invites the possibility of selectively targeting the life cycle of P. falciparum in order to prevent cerebral malaria. PfPK5 bears a high degree of sequence identity (>58%) to a structurally conserved family of mammalian kinases known as the cyclin-dependent kinases (CDKs). The CDKs are the key regulatory elements governing the ordered progression of the mammalian cell cycle. With numerous X-ray crystal structures of CDK2 to provide a structural template, here we present a three-dimensional structural model of PfPK5 constructed using computer-based homology modeling techniques. Our model was used to compare the ATP binding site of PfPK5 with that of the mammalian kinase CDK2. Furthermore, kinase-ligand interactions of PfPK5 with known inhibitors were investigated and compared to available crystal structures of CDK2 with inhibitors bound. The focus of the study is to identify similarities and differences between the ATP binding sites of the two kinases that can be exploited for future rational drug design.     

PKR-inhibitor binds efficiently with human microtubule affinity-regulating kinase 4

MAP/microtubule affinity-regulating kinase 4 (MARK4) plays a central role in the cellular physiology, and it is inseparably linked with many human diseases including cancer, diet induced obesity, type2 diabetes and neurodegenerative disorders. Here, we studied the interaction of PKR-inhibitor with two variants of human MARK4. One variant is named as MARK4-F1 which has 59 N-terminal residues along with kinase domain while another variant is MARK4-F2 which has kinase domain only. Molecular-docking, molecular dynamics (MD) simulation and fluorescence-binding studies were undertaken to understand the role of N-terminal 59-residues in the binding of substrate/inhibitors. Molecular docking studies revealed that the PKR-inhibitor binds in the large hydrophobic cavity of the kinase domain of MARK4 through several hydrophobic and hydrogen-bonded interactions. Furthermore, MD simulation showed a stable parameters for the complexes of both MARK4-F1 and MARK4-F2 to PKR-inhibitor with marginal difference in their binding affinities. A significant decrease in the fluorescence intensity of MARK4 was observed on successive addition of the PKR-inhibitor. Using fluorescence data we have calculated the binding-affinity and the number of binding site of PKR-inhibitor to the MARK4. A significantly high binding affinity was observed for the PKR-inhibitor to the MARK4 variants. However, there is no any significant difference in the binding affinity of PKR-inhibitor to the MARK4 variants was observed, indicating that 59 N-terminal residues of MARK4-F1 are not playing a crucial role in the ligand binding. The present study will provide an insights into designing of new PKR-inhibitor derivative as potent and selective therapeutic agent against many life threatening diseases which are associated with MARK4.     

SAHA-based novel HDAC inhibitor design by core hopping method

The catalytic activity of the histone deacetylase (HDAC) is directly relevant to the pathogenesis of cancer, and HDAC inhibitors represented a promising strategy for cancer therapy. SAHA (suberoanilide hydroxamic acid), an effective HDAC inhibitor, is an anti-cancer agent against T-cell lymphoma. However, SAHA has adverse effects such as poor pharmacokinetic properties and severe toxicities in clinical use. In order to identify better HDAC inhibitors, a compound database was established by core hopping of SAHA, which was then docked into HDAC-8 (PDB ID: 1T69) active site to select a number of candidates with higher docking score and better interaction with catalytic zinc ion. Further ADMET prediction was done to give ten compounds. Molecular dynamics simulation of the representative compound 101 was performed to study the stability of HDAC8-inhibitor system. This work provided an approach to design novel high-efficiency HDAC inhibitors with better ADMET properties.     

Computational determination of binding structures and free energies of glucose 6-phosphate dehydrogenase with novel steroid inhibitors

Glucose 6-phosphate dehydrogenase (G6PD), the first and the rate-limiting enzyme in the pentose phosphate pathway (PPP), catalyzes the oxidation of G6P to 6-phosphogluconolactone and the reduction of NADP⁺ to NADPH. Its key role in cancer promotes the development of a potent and selective inhibitor that might increase cancer cell death when combined with radiotherapy. In the present study, we investigated the detailed binding modes and binding free energies for G6PD interacting with a promising series of recently developed inhibitors, i.e., the steroid derivatives, by performing molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. The docking indicates that the inhibitors occupy the binding sites of both G6P and NADP⁺. The calculated binding free energies on the basis of the MD-simulated enzyme–inhibitor complexes are in good agreement with the experimental activity data for all of the examined inhibitors. The valuable insights into the detailed enzyme–inhibitor binding including the important intermolecular interactions, e.g., the hydrogen bond interaction and the hydrophobic interaction, have been provided. The computational results provide new insights into future rational design of more potent inhibitors of G6PD as a treatment for cancer.     

Binding mechanism of CDK5 with roscovitine derivatives based on molecular dynamics simulations and MM/PBSA methods

Roscovitine derivatives are potent inhibitors of cyclin-dependent kinase 5 (CDK5), but they exhibit different activities, which has not been understood clearly up to now. On the other hand, the task of drug design is difficult because of the fuzzy binding mechanism. In this context, the methods of molecular docking, molecular dynamics (MD) simulation, and binding free energy analysis are applied to investigate and reveal the detailed binding mechanism of four roscovitine derivatives with CDK5. The electrostatic and van der Waals interactions of the four inhibitors with CDK5 are analyzed and discussed. The calculated binding free energies in terms of MM-PBSA method are consistent with experimental ranking of inhibitor effectiveness for the four inhibitors. The hydrogen bonds of the inhibitors with Cys83 and Lys33 can stabilize the inhibitors in binding sites. The van der Waals interactions, especially the pivotal contacts with Ile10 and Leu133 have larger contributions to the binding free energy and play critical roles in distinguishing the variant bioactivity of four inhibitors. In terms of binding mechanism of the four inhibitors with CDK5 and energy contribution of fragments of each inhibitor, two new CDK5 inhibitors are designed and have stronger inhibitory potency.     

Unravelling the structural interactions between PKR kinase domain and its small molecule inhibitors using computational approaches

The RNA-dependent protein kinase (PKR), an eIF2α kinase plays an important role in anti-viral response, apoptosis and cell survival. It is also implicated to play a role in several cancers, metabolic and neurodegenerative disorders. A few ATP competitive inhibitors of the PKR have been reported in the literature with promising results in vitro and in vivo. The aim of this study was to unravel the structural interactions between these inhibitors and the PKR kinase domain using molecular simulations and docking. Our study reveals that the reported inhibitors bind in the adenine pocket and form hydrogen bonds with the hinge region and vdW interactions with non-polar residues in the binding site. The most potent inhibitor has several favorable interactions with the binding site and induces the P-loop to fold inward, creating a significant hydrophobic enclosure for itself. The computed binding free energies of these inhibitors are in accord with experimental data (IC₅₀). Strategies to design potent and selective PKR inhibitors are discussed to overcome the reported promiscuity.     

Homology modelling,docking, pharmacophore and site directed mutagenesis analysis to identify the critical amino acid residue of PknI from Mycobacterium tuberculosis

Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen. PknI is one of the 11 functional Serine/Threonine Protein Kinases which is predicted to regulate the cell division of M. tuberculosis. In order to find newer drugs and vaccine we need to understand the pathogenesis of the disease. We have used the bioinformatics approach to identify the functionally active residues of PknI and to confirm the same with wet lab experiments. In the current study, we have created homology model for PknI and have done comparative structural analysis of PknI with other kinases. Molecular docking studies were done with a library of kinase inhibitors and T95 was found as the potent inhibitor for PknI. Based on structure based pharmacophore analysis of kinase substrate complexes, Lys 41 along with Asp90, Val92 and Asp96 were identified as functionally important residues. Further, we used site directed mutagenesis technique to mutate Lys 41 to Met resulting in defective cell division of Mycobacterium smegmatis mc². Overall, the proposed model together with its binding features gained from pharmacophore docking studies helped in identifying ligand inhibitor specific to PknI which was confirmed by laboratory experiments.