Putative Role of Cholesterol in Shaping the Structural and Functional Dynamics of Smoothened (SMO).

The smoothened (SMO) receptor belongs to the superfamily of class F G protein-coupled receptors (GPCRs) and is a potential drug target in several types of cancer. It has two ligand binding sites, respectively, in the cysteine-rich domain (CRD) and the transmembrane domain (TMD). It has been shown that cholesterol is important for its activation and function. However, the molecular-level understanding of SMO dynamics in the presence of cholesterol has not been explored in sufficient detail. In this work, we have carried out atomistic molecular dynamics simulations totaling 3.6 µs to analyze the effect of cholesterol binding to TMD and/or CRD on the structure and dynamics of the SMO receptor. Our results show that the presence of cholesterol in the CRD and TMD, respectively, alters the conformational dynamics of SMO differently. We reported that the reorganization of the D-R-E network at the extracellular end of the TMD is important for the high activity of SMO. In general, the transmembrane helices 5, 6, and 7 and helix 8 are most affected, which, in turn, leads to changes in the CRD and intracellular cytoplasmic domain (ICD) dynamics patterns depending on the presence or absence of cholesterol in the CRD and/or the TMD. We have also reported that the interaction of membrane lipids with SMO is different in different SMO states. These results agree with the experimental structural observations and data of cholesterol-bound and unbound structures of SMO and add to our molecular understanding of the SMO-cholesterol interaction.

De novo design of anti-tuberculosis agents using a structure-based deep learning method.

Mycobacterium tuberculosis (Mtb) is a pathogen of major concern due to its ability to withstand both first- and second-line antibiotics, leading to drug resistance. Thus, there is a critical need for identification of novel anti-tuberculosis agents targeting Mtb-specific proteins. The ceaseless search for novel antimicrobial agents to combat drug-resistant bacteria can be accelerated by the development of advanced deep learning methods, to explore both existing and uncharted regions of the chemical space. The adaptation of deep learning methods to under-explored pathogens such as Mtb is a challenging aspect, as most of the existing methods rely on the availability of sufficient target-specific ligand data to design novel small molecules with optimized bioactivity. In this work, we report the design of novel anti-tuberculosis agents targeting the Mtb chorismate mutase protein using a structure-based drug design algorithm. The structure-based deep learning method relies on the knowledge of the target protein's binding site structure alone for conditional generation of novel small molecules. The method eliminates the need for curation of a high-quality target-specific small molecule dataset, which remains a challenge even for many druggable targets, including Mtb chorismate mutase. Novel molecules are proposed, that show high complementarity to the target binding site. The graph attention model could identify the probable key binding site residues, which influenced the conditional molecule generator to design new molecules with pharmacophoric features similar to the known inhibitors.

Sonochemical synthesis and biological evaluation of isoquinolin-1(2H)-one/isoindolin-1-one derivatives: Discovery of a positive ago-allosteric modulator (PAAM) of 5HT2CR.

Efforts have been devoted for the discovery and development of positive allosteric modulators (PAMs) of 5-HT2CR because of their potential advantages over the orthosteric agonist like Lorcaserin that was withdrawn from the market. On the other hand, pursuing a positive ago-allosteric modulator (PAAM) is considered as beneficial particularly when an agonist is not capable of affecting the potency of the endogenous agonist sufficiently. In search of a suitable PAAM of 5-HT2CR we adopted an in silico based approach that indicated the potential of the 3-(1-hydroxycycloalkyl) substituted isoquinolin-1-one derivatives against the 5-HT2CR as majority of these molecules interacted with the site other than that of Lorcaserin with superior docking scores. These compounds along with the regioisomeric 3-methyleneisoindolin-1-one derivatives were prepared via the Cu(OAc)2 catalyzed coupling of 2-iodobenzamide with 1-ethynylcycloalkanol under ultrasound irradiation. According to the in vitro studies, most of these compounds were not only found to be potent and selective agonists but also emerged as PAAM of 5-HT2CR whereas Lorcaserin did not show PAAM activities. According to the SAR study the isoquinolin-1(2H)-ones appeared as better PAAM than isoindolin-1-ones whereas the presence of hydroxyl group appeared to be crucial for the activity. With the potent PAAM activity for 5-HT2CR (EC50 = 1 nM) and 107 and 86-fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B the compound 4i was identified as a hit molecule. The compound showed good stability in male BALB/c mice brain homogenate (∼85 % remaining after 2 h), moderate stability in the presence of rat liver microsomes (42 % remaining after 1 h) and acceptable PK properties with fast reaching in the brain maintaining âˆ¼ 1:1 brain/plasma concentration ratio. The compound at a dose of 50 mg/kg exhibited decreased trend in the food intake starting from day 3 in S.D. rats, which reached significant by 5th day, and the effect was comparable to Lorcaserin (10 mg/kg) on day 5. Thus, being the first example of PAAM of 5-HT2CR the compound 4i is of further medicinal interest.

Structural dynamics of Smoothened (SMO) in the ciliary membrane and its interaction with membrane lipids.

The Smoothened receptor (SMO, a 7 pass transmembrane domain, Class F GPCR family protein) plays a crucial role in the Hedgehog (HH) signaling pathway, which is involved in embryonic development and is implicated in various types of cancer throughout the animal kingdom. In the absence of HH signaling, SMO is inhibited by Patched 1 (PTC1; a 12 pass transmembrane domain protein), which is localized in the primary cilia. HH binding leads to the dislocation of PTC1 from the cilia, thus making way for SMO to localize in the primary cilia, as an essential prerequisite for its activation. We have carried out MARTINI coarse-grained molecular dynamics simulations of SMO in POPC and in ciliary membrane models, respectively, to study the interactions of SMO with cholesterol and other lipid molecules in the ciliary membrane, and to gain molecular-level insights into the role of the primary cilia in shaping the functional dynamics of SMO. We are able to identify the interaction of membrane cholesterols with definite sites and domains within SMO and relate them with known cholesterol-binding sequence and structure motifs. We show that cholesterol interactions with the transmembrane domain TMD, unlike those with the cysteine-rich domain (CRD) and the intracellular domain (ICD), are through residues belonging to known cholesterol-binding motifs. Notably, a few persistent interactions of cholesterol with lower TM cholesterol-binding domains are governed by the presence of multiple cholesterol-binding motifs. These analyses have also helped to identify and define a strict cholesterol consensus motif (CCM), which may well steer cholesterol into the hitherto identified binding sites within the TMD of SMO. We have also reported the interaction of phosphatidylinositol 4-phosphate with the intracellular region of transmembrane (TM) helices (TM1, TM3, TM4, and TM5), intracellular loop1, helix8, and Arg/Lys clusters of the ICD. Structural analysis of SMO domains shows significant changes in the CRD and ICD, during the course of the simulation. Further detailed analysis of the dynamics of the TMD reveals the movements of TM5, TM6, and TM7, linked with the helix8, which are possibly involved in shaping the conformational disposition of the ICD. The movement of these TM helices could possibly be a consequence of interactions involving the extracellular domain and extracellular loops. In addition, our analysis also shows that phosphatidylinositol-4-phosphate (PI4P), along with some ICD cholesterols, are implicated in anchoring SMO in the membrane.

De Novo Structure-Based Drug Design Using Deep Learning.

In recent years, deep learning-based methods have emerged as promising tools for de novo drug design. Most of these methods are ligand-based, where an initial target-specific ligand data set is necessary to design potent molecules with optimized properties. Although there have been attempts to develop alternative ways to design target-specific ligand data sets, availability of such data sets remains a challenge while designing molecules against novel target proteins. In this work, we propose a deep learning-based method, where the knowledge of the active site structure of the target protein is sufficient to design new molecules. First, a graph attention model was used to learn the structure and features of the amino acids in the active site of proteins that are experimentally known to form protein-ligand complexes. Next, the learned active site features were used along with a pretrained generative model for conditional generation of new molecules. A bioactivity prediction model was then used in a reinforcement learning framework to optimize the conditional generative model. We validated our method against two well-studied proteins, Janus kinase 2 (JAK2) and dopamine receptor D2 (DRD2), where we produce molecules similar to the known inhibitors. The graph attention model could identify the probable key active site residues, which influenced the conditional molecule generator to design new molecules with pharmacophoric features similar to the known inhibitors.

Network-Based Analysis of Fatal Comorbidities of COVID-19 and Potential Therapeutics.

COVID-19 is a highly contagious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The case-fatality rate is significantly higher in older patients and those with diabetes, cancer or cardiovascular disorders. The human proteins, angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2) and basigin (BSG), are involved in high-confidence host-pathogen interactions with SARS-CoV-2 proteins. We considered these three proteins as seed nodes and applied the random walk with restart method on the human interactome to construct a protein-protein interaction sub-network, which captures the effects of viral invasion. We found that 'Insulin resistance', 'AGE-RAGE signaling in diabetic complications' and 'adipocytokine signaling' were the common pathways associated with diabetes, cancer and cardiovascular disorders. The association of these critical pathways with aging and its related diseases explains the molecular basis of COVID-19 fatality. We further identified drugs that have effects on these proteins/pathways based on gene expression studies. We particularly focused on drugs that significantly downregulate ACE2 along with other critical proteins identified by the network-based approach. Among them, COL-3 had earlier shown activity against acute lung injury and acute respiratory distress, while entinostat and mocetinostat have been investigated for non-small-cell lung cancer. We propose that these drugs can be repurposed for COVID-19.

De novo design of new chemical entities for SARS-CoV-2 using artificial intelligence.

Background: The novel coronavirus SARS-CoV-2 has severely affected the health and economy of several countries. Multiple studies are in progress to design novel therapeutics against the potential target proteins in SARS-CoV-2, including 3CL protease, an essential protein for virus replication. Materials & methods: In this study we employed deep neural network-based generative and predictive models for de novo design of small molecules capable of inhibiting the 3CL protease. The generative model was optimized using transfer learning and reinforcement learning to focus around the chemical space corresponding to the protease inhibitors. Multiple physicochemical property filters and virtual screening score were used for the final screening. Conclusion: We have identified 33 potential compounds as ideal candidates for further synthesis and testing against SARS-CoV-2.

Accelerating De Novo Drug Design against Novel Proteins Using Deep Learning.

In the world plagued by the emergence of new diseases, it is essential that we accelerate the drug design process to develop new therapeutics against them. In recent years, deep learning-based methods have shown some success in ligand-based drug design. Yet, these methods face the problem of data scarcity while designing drugs against a novel target. In this work, the potential of deep learning and molecular modeling approaches was leveraged to develop a drug design pipeline, which can be useful for cases where there is limited or no availability of target-specific ligand datasets. Inhibitors of the homologues of the target protein were screened at the active site of the target protein to create an initial target-specific dataset. Transfer learning was used to learn the features of the target-specific dataset. A deep predictive model was utilized to predict the docking scores of newly designed molecules. Both these models were combined using reinforcement learning to design new chemical entities with an optimized docking score. The pipeline was validated by designing inhibitors against the human JAK2 protein, where none of the existing JAK2 inhibitors were used for training. The ability of the method to reproduce existing molecules from the validation dataset and design molecules with better binding energy demonstrates the potential of the proposed approach.

Elucidation of an essential function of the unique charged domain of Plasmodium topoisomerase III.

Topoisomerase III (TopoIII) along with RecQ helicases are required for the resolution of abnormal DNA structures that result from the stalling of replication forks. Sequence analyses have identified a putative TopoIII in the Plasmodium falciparum genome (PfTopoIII). PfTopoIII shows dual nuclear and mitochondrial localization. The expression and association of PfTopoIII with mtDNA is tightly linked to the asexual replication of the parasite. In this study, we observed that PfTopoIII physically interacts with PfBlm and PfWrn. Sequence alignment and domain analyses have revealed that it contains a unique positively charged region, spanning 85 amino acids, within domain II. A molecular dynamics simulation study revealed that this unstructured domain communicates with DNA and attains a thermodynamically stable state upon DNA binding. Here, we found that the association between PfTopoIII and the mitochondrial genome is negatively affected by the absence of the charged domain. Our study shows that PfTOPOIII can completely rescue the slow growth phenotype of the ΔtopoIII strain in Saccharomyces cerevisiae, but neither PfY421FtopoIII (catalytic-active site mutant) nor Pf(Δ259-337)topoIII (charged region deletion mutant) can functionally complement ScTOPOIII. Hydroxyurea (HU) led to stalling of the replication fork during the S phase, caused moderate toxicity to the growth of P. falciparum, and was associated with concomitant transcriptional upregulation of PfTOPOIII. In addition, ectopic expression of PfTOPOIII reversed HU-induced toxicity. Interestingly, the expression of Pf(Δ259-337)topoIII failed to reverse HU-mediated toxicity. Taken together, our results establish the importance of TopoIII during Plasmodium replication and emphasize the essential requirement of the charged domain in PfTopoIII function.

Elucidation of DNA Repair Function of PfBlm and Potentiation of Artemisinin Action by a Small-Molecule Inhibitor of RecQ Helicase.

Artemisinin (ART)-based combination therapies are recommended as first- and second-line treatments for Plasmodium falciparum malaria. Here, we investigated the impact of the RecQ inhibitor ML216 on the repair of ART-mediated damage in the genome of P. falciparumPfBLM and PfWRN were identified as members of the RecQ helicase family in P. falciparum However, the role of these RecQ helicases in DNA double-strand break (DSB) repair in this parasite has not been explored. Here, we provide several lines of evidence to establish the involvement of PfBlm in DSB repair in P. falciparum First, we demonstrate that PfBlm interacts with two well-characterized DSB repair proteins of this parasite, namely, PfRad51 and PfalMre11. Second, we found that PfBLM expression was upregulated in response to DNA-damaging agents. Third, through yeast complementation studies, we demonstrated that PfBLM could complement the DNA damage sensitivity of a Δsgs1 mutant of Saccharomyces cerevisiae, in contrast to the helicase-dead mutant PfblmK83R Finally, we observe that the overexpression of PfBLM induces resistance to DNA-damaging agents and offers a survival advantage to the parasites. Most importantly, we found that the RecQ inhibitor ML216 inhibits the repair of DSBs and thereby renders parasites more sensitive to ART. Such synergism between ART and ML216 actions was observed for both drug-sensitive and multidrug-resistant strains of P. falciparum Taken together, these findings establish the implications of PfBlm in the Plasmodium DSB repair pathway and provide insights into the antiparasitic activity of the ART-ML216 combination.IMPORTANCE Malaria continues to be a serious threat to humankind not only because of the morbidity and mortality associated with the disease but also due to the huge economic burden that it imparts. Resistance to all available drugs and the unavailability of an effective vaccine cry for an urgent discovery of newer drug targets. Here, we uncovered a role of the PfBlm helicase in Plasmodium DNA double-strand break repair and established that the parasitic DNA repair mechanism can be targeted to curb malaria. The small-molecule inhibitor of PfBlm tested in this study acts synergistically with two first-line malaria drugs, artemisinin (ART) and chloroquine, in both drug-sensitive and multidrug-resistant strains of P. falciparum, thus qualifying this chemical as a potential partner in ART-based combination therapy. Additionally, the identification of this new specific inhibitor of the Plasmodium homologous recombination (HR) mechanism will now allow us to investigate the role of HR in Plasmodium biology.

Network analysis of hydroxymethylbilane synthase dynamics.

Hydroxymethylbilane synthase (HMBS) is one of the key enzymes of the heme biosynthetic pathway that catalyzes porphobilinogen to form the linear tetrapyrrole 1-hydroxymethylbilane through four intermediate steps. Mutations in the human HMBS (hHMBS) can lead to acute intermittent porphyria (AIP), a lethal metabolic disorder. The molecular basis of importance of the amino acid residues at the catalytic site of hHMBS has been well studied. However, the role of non-active site residues toward the activity of the enzyme and hence the association of their mutations with AIP is not known. Network-based analyses of protein structures provide a systems approach to understand the correlations of the residues through a series of inter-residue interactions. We analyzed the dynamic network representation of HMBS protein derived from five molecular dynamics trajectories corresponding to the five steps of pyrrole polymerization. We analyzed the network clusters for each stage and identified the amino acid residues and interactions responsible for the structural stability and catalytic function of the protein. The analysis of high betweenness nodes and interaction paths from the active site help in understanding the molecular basis of the effect of non-active site AIP-causing mutations on the catalytic activity.

Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release.

5'-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.

A network-based approach reveals novel invasion and Maurer's clefts-related proteins in Plasmodium falciparum.

Malaria continues to be a major concern in developing countries despite continuous efforts to find a cure for the disease. Understanding the pathogenesis mechanism is necessary to identify more effective drug targets against malaria. Many years of experimental research have generated a large amount of data for the malarial parasite, Plasmodium falciparum. These data are useful to understand the importance of certain parasite proteins, but it often remains unclear how these proteins come together, interact with other proteins and carry out their function. Identification of all proteins involved in pathogenesis is an important step towards understanding the molecular mechanism of pathogenesis. In this study, dynamic stage-specific protein-protein interaction networks were created based on gene expression data during the parasite's intra-erythrocytic stages and static protein-protein interaction data. Using previously known proteins of a biological event as seed proteins, the random walk with restart (RWR) method was used on the dynamic protein-protein interaction networks to identify novel proteins related to that event. Two screening procedures namely, permutation test and GO enrichment test were performed to increase the reliability of the RWR predictions. The proposed method was first validated on Plasmodium falciparum proteins related to invasion, where it could reproduce the existing knowledge from a small set of seed proteins. It was then used to identify novel Maurer's clefts resident proteins, where it could identify 152 parasite proteins. We show that the current approach can annotate conserved proteins with unknown function. The predicted proteins can help build a mechanistic model for disease pathogenesis, which will be useful in identifying new drug targets.

Chitooligosaccharide binding to CIA17 (Coccinia indica agglutinin). Thermodynamic characterization and formation of higher order complexes.

CIA17 is a PP2-like, homodimeric lectin made up of 17 kDa subunits present in the phloem exudate of ivy gourd (Coccinia indica). Isothermal titration calorimetric (ITC) studies on the interaction of chitooligosaccharides [(GlcNAc)2-6] showed that the dimeric protein has two sugar binding sites which recognize chitotriose with ~70-fold higher affinity than chitobiose, indicating that the binding site is extended in nature. ITC, atomic force microscopic and non-denaturing gel electrophoresis studies revealed that the high-affinity interaction of CIA17 with chitohexaose (Ka = 1.8 × 107 M-1) promotes the formation of protein oligomers. Computational studies involving homology modeling, molecular docking and molecular dynamics simulations on the binding of chitooligosaccharides to CIA17 showed that the protein binding pocket accommodates up to three GlcNAc residues. Interestingly, docking studies with chitohexaose indicated that its two triose units could interact with binding sites on two protein molecules to yield dimeric complexes of the type CIA17-(GlcNAc)6-CIA17, which can extend in length by the binding of additional chitohexaose and CIA17 molecules. These results suggest that PP2 proteins play a role in plant defense against insect/pathogen attack by directly binding with the higher chain length chitooligosaccharides and forming extended, filamentous structures, which facilitate wound sealing.

Unfolding Transitions of Peripheral Subunit Binding Domains Show Cooperative Behavior.

Characterization of native, intermediate, and denatured states is crucial for understanding the factors influencing the stability of proteins. We have carried out molecular dynamics simulations to study the unfolding of three peripheral subunit binding domains (PSBDs): E. coli BBL, Bacillus stearothermophilus E3BD, and human hbSBD, at three different temperatures: 300, 330, and 400 K, and in the presence of two solvents: water and 5 M guanidinium hydrochloride (GndCl) solution. These proteins share similar folds, with two parallel helices, maintained via a hydrophobic core comprising residues from their interconnecting loop. BBL is more sensitive to thermal and chemical denaturation in comparison to hbSBD, and E3BD is the most stable of all of the three proteins. The effect of temperature on the stability of these proteins is more pronounced in "water-only" simulations compared to that in the presence of guanidium hydrochloride in high concentrations. Our results show cooperative unfolding transitions of these proteins, which are triggered by an initial melting of the C-terminal helix H2. The consequent loss of interhelical interactions or native contacts, as observed, leads to the subsequent melting of the N-terminal helix H1.

Computational modeling of the catalytic mechanism of hydroxymethylbilane synthase.

Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthesis pathway, catalyzes the formation of 1-hydroxymethylbilane (HMB) by a stepwise polymerization of four molecules of porphobilinogen (PBG) using the dipyrromethane (DPM) cofactor. The mechanism by which HMBS polymerizes four units of PBG has not been elucidated to date. In vitro and in silico studies on HMBS have suggested certain residues with catalytic importance, but their specific role in the catalysis is unclear. To understand the catalytic mechanism of HMBS, quantum mechanical (QM) calculations were performed on model systems obtained from the active site of the human HMBS enzyme. The addition of one molecule of PBG to the DPM cofactor is carried out in four steps: (1) protonation of the substrate, PBG; (2) deamination of PBG; (3) electrophilic addition of the deaminated substrate to the terminal pyrrole ring of the enzyme-bound DPM cofactor and (4) deprotonation of the carbon atom at the α-position of the second ring of DPM. Based on the energy profiles from the QM calculations on cluster models, R26 is proposed to be the best suitable proton donor to the PBG moiety, which aids in the deamination of the substrate. During the electrophilic addition step, the intermediate formed is stabilized by the carboxylate side chain of the D99 residue. In the final deprotonation step, an extra proton from the second ring of DPM is transferred to R26 via the carboxylate side chain of D99, thus completing one cycle of the catalytic mechanism. The residues in the cluster model seem to play an important role in obtaining accurate energy barriers. All the stationary points along the reaction pathway have been characterized using QM calculations. The rate limiting step for the complete mechanism is found to be the deamination of the PBG moiety. The results of this study provide a detailed understanding of the catalytic mechanism and would help design future studies aimed at modulating the activity of HMBS.

TPP riboswitch aptamer: Role of Mg2+ ions, ligand unbinding, and allostery.

Riboswitches are non-coding RNAs that regulate gene expression in response to the binding of metabolites. Their abundance in bacteria makes them ideal drug targets. The prokaryotic thiamine pyrophosphate (TPP) riboswitch regulates gene expression in a wide range of bacteria by undergoing conformational changes in response to the binding of TPP. Although an experimental structure for the aptamer domain of the riboswitch is now available, details of the conformational changes that occur during the binding of the ligand, and the factors that govern these conformational changes, are still not clear. This study employs microsecond-scale molecular dynamics simulations to provide insights into the functioning of the riboswitch aptamer in atomistic detail. A mechanism for the transmission of conformational changes from the ligand-binding site to the P1 switch helix is proposed. Mg2+ ions in the binding site play a critical role in anchoring the ligand to the riboswitch. Finally, modeling the egress of TPP from the binding site reveals a two-step mechanism for TPP unbinding. Findings from this study can motivate the design of future studies aimed at modulating the activity of this drug target.

2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK.

AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model.

Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP.

Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human HMBS (hHMBS) cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme's domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP.

A novel SIRT1 inhibitor, 4bb induces apoptosis in HCT116 human colon carcinoma cells partially by activating p53.

The NAD+-dependent protein deacetylase SIRT1 has emerged as an important target for epigenetic therapeutics of colon cancer as its increased expression is associated with cancer progression. Additionally, SIRT1 represses p53 function via deacetylation, promoting tumor growth. Therefore, inhibition of SIRT1 is of great therapeutic interest for the treatment of colon cancer. Here, we report discovery of a novel quinoxaline based small molecule inhibitor of human SIRT1, 4bb, investigated its effect on viability of colon cancer cells and molecular mechanism of action. In vitro, 4bb is a significantly more potent SIRT1 inhibitor, compared to ß-naphthols such as sirtinol, cambinol. Increasing concentration of 4bb decrease viability of colon cancer cells but, does not affect the viability of normal dermal fibroblasts depicting cancer cell specificity. Further, 4bb treatment increased p53 acetylation, Bax expression and induced caspase 3 cleavage suggesting that the death of HCT116 colon cancer cells occur through intrinsic pathway of apoptosis. Overall, our results presents 4bb as a new class of human SIRT1 inhibitor and suggest that inhibition of SIRT1 by 4bb induces apoptosis of colon cancer cells at least in part via activating p53 by preventing p53 deacetylation, increasing Bax expression and inducing caspases. Therefore, this molecule provide an opportunity for lead optimization and may help in development of novel, non-toxic epigenetic therapeutics for colon cancer.