Discovery of Novel Liver-Stage Antimalarials through Quantum Similarity.

Without quantum theory any understanding of molecular interactions is incomplete. In principal, chemistry, and even biology, can be fully derived from non-relativistic quantum mechanics. In practice, conventional quantum chemical calculations are computationally too intensive and time consuming to be useful for drug discovery on more than a limited basis. A previously described, original, quantum-based computational process for drug discovery and design bridges this gap between theory and practice, and allows the application of quantum methods to large-scale in silico identification of active compounds. Here, we show the results of this quantum-similarity approach applied to the discovery of novel liver-stage antimalarials. Testing of only five of the model-predicted compounds in vitro and in vivo hepatic stage drug inhibition assays with P. berghei identified four novel chemical structures representing three separate quantum classes of liver-stage antimalarials. All four compounds inhibited liver-stage Plasmodium as a single oral dose in the quantitative PCR mouse liver-stage sporozoites-challenge model. One of the newly identified compounds, cethromycin [ABT-773], a macrolide-quinoline hybrid, is a drug with an extensive (over 5,000 people) safety profile warranting its exploitation as a new weapon for the current effort of malaria eradication. The results of our molecular modeling exceed current state-of-the-art computational methods. Drug discovery through quantum similarity is data-driven, agnostic to any particular target or disease process that can evaluate multiple phenotypic, target-specific, or co-crystal structural data. This allows the incorporation of additional pharmacological requirements, as well as rapid exploration of novel chemical spaces for therapeutic applications.

Discovery of potent, novel Nrf2 inducers via quantum modeling, virtual screening, and in vitro experimental validation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH-associating protein 1 (Keap1). To identify novel small molecules that will disturb Nrf2-Keap1 binding and promote activation of the Nrf2- antioxidant response element pathway, we generated a quantum model based on the structures of known Nrf2- antioxidant response element activators. We used the quantum model to perform in silico screening on over 18 million commercially available chemicals to identify the structures predicted to activate the Nrf2- antioxidant response element pathway based on the quantum model. The top hits were tested in vitro, and half of the predicted hits activated the Nrf2-antioxidant response element pathway significantly in primary cell culture. In addition, we identified a new family of Nrf2-antioxidant response element-activating structures that all have comparable activity to tBHQ and protect against oxidative stress and dopaminergic toxins in vitro. The improved ability to identify potent activators of Nrf2 through the combination of in silico and in vitro screening described here improves the speed and cost associated with screening Nrf2-antioxidant response element -activating compounds for drug development.

Discovery of potent, novel, non-toxic anti-malarial compounds via quantum modelling, virtual screening and in vitro experimental validation.

BACKGROUND: Developing resistance towards existing anti-malarial therapies emphasize the urgent need for new therapeutic options. Additionally, many malaria drugs in use today have high toxicity and low therapeutic indices. Gradient Biomodeling, LLC has developed a quantum-model search technology that uses quantum similarity and does not depend explicitly on chemical structure, as molecules are rigorously described in fundamental quantum attributes related to individual pharmacological properties. Therapeutic activity, as well as toxicity and other essential properties can be analysed and optimized simultaneously, independently of one another. Such methodology is suitable for a search of novel, non-toxic, active anti-malarial compounds. METHODS: A set of innovative algorithms is used for the fast calculation and interpretation of electron-density attributes of molecular structures at the quantum level for rapid discovery of prospective pharmaceuticals. Potency and efficacy, as well as additional physicochemical, metabolic, pharmacokinetic, safety, permeability and other properties were characterized by the procedure. Once quantum models are developed and experimentally validated, the methodology provides a straightforward implementation for lead discovery, compound optimizzation and de novo molecular design. RESULTS: Starting with a diverse training set of 26 well-known anti-malarial agents combined with 1730 moderately active and inactive molecules, novel compounds that have strong anti-malarial activity, low cytotoxicity and structural dissimilarity from the training set were discovered and experimentally validated. Twelve compounds were identified in silico and tested in vitro; eight of them showed anti-malarial activity (IC50 ≤ 10 µM), with six being very effective (IC50 ≤ 1 µM), and four exhibiting low nanomolar potency. The most active compounds were also tested for mammalian cytotoxicity and found to be non-toxic, with a therapeutic index of more than 6,900 for the most active compound. CONCLUSIONS: Gradient's metric modelling approach and electron-density molecular representations can be powerful tools in the discovery and design of novel anti-malarial compounds. Since the quantum models are agnostic of the particular biological target, the technology can account for different mechanisms of action and be used for de novo design of small molecules with activity against not only the asexual phase of the malaria parasite, but also against the liver stage of the parasite development, which may lead to true causal prophylaxis.

BAAV transcytosis requires an interaction with beta-1-4 linked- glucosamine and gp96.

Cell surface carbohydrates play an important role in virus entry and intracellular trafficking. Bovine Adeno-Associated Virus (BAAV) uses plasma membrane gangliosides for transduction and infection. In addition, independent of the infectious pathway, BAAV also has the ability to pass through barrier epithelia and endothelia using a transcytosis pathway dependent upon the presence of cell surface carbohydrates. Thus, in order to better define the carbohydrate interactions that are necessary for BAAV infection or transcytosis, a glycan microarray composed of both natural and synthetic carbohydrates was probed with HA-tagged BAAV particles. This identified chitotriose, a trimer of beta-1-4-linked N-acetyl glucosamine, as having an interaction with BAAV. Competition experiments showed that the BAAV interaction with this carbohydrate is not necessary for infection but is instead important in the transcytosis pathway. The beta-1-4-linked N-acetyl glucosamine modification has been reported on gp96, a glycoprotein involved in the transcytosis of bacteria and toxins. Significantly, immunoprecipitation and competition experiments with an anti-gp96 antibody and a soluble form of gp96, respectively, showed this glycoprotein can also interact with BAAV to serve as a receptor for its transcytosis.

Rho GTPases modulate entry of Ebola virus and vesicular stomatitis virus pseudotyped vectors.

To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.

Structurally mapping the diverse phenotype of adeno-associated virus serotype 4.

The adeno-associated viruses (AAVs) can package and deliver foreign DNA into cells for corrective gene delivery applications. The AAV serotypes have distinct cell binding, transduction, and antigenic characteristics that have been shown to be dictated by the capsid viral protein (VP) sequence. To understand the contribution of capsid structure to these properties, we have determined the crystal structure of AAV serotype 4 (AAV4), one of the most diverse serotypes with respect to capsid protein sequence and antigenic reactivity. Structural comparison of AAV4 to AAV2 shows conservation of the core beta strands (betaB to betaI) and helical (alphaA) secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations (I to IX), some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. These include AAV4 having a deeper twofold depression, wider and rounder protrusions surrounding the threefold axes, and a different topology at the top of the fivefold channel from that of AAV2. Also, the previously observed "valleys" between the threefold protrusions, containing AAV2's heparin binding residues, are narrower in AAV4. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes.

Structure of adeno-associated virus type 4.

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-A resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology.

Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 4.

Adeno-associated virus (AAV) serotypes 1 to 5 are currently under development as clinical gene delivery vectors for the treatment of human diseases. However, the ubiquitous nature of their cell surface receptors, heparin sulfate (AAV2 and 3) and sialic acids (AAV4 and 5), can preclude specific tissue targeting in vivo. Structural studies of AAV4 were initiated to characterize its capsid surface for re-targeting manipulations. Crystals obtained diffracted synchrotron radiation to 3.2 A resolution. The unit cell is body-centered orthorhombic, I222, with a = 339.6, b = 319.2 and c = 285.0 A. The virus particle orientation and position have been determined.

Scalable purification of adeno-associated virus type 2, 4, or 5 using ion-exchange chromatography.

The availability of high-titer, high-purity, adeno-associated virus type 2 (AAV2) stocks has dramatically increased our understanding of this virus and its utility as a gene transfer vector. Current methods of purification take advantage of the stable interaction of AAV2 with heparin sulfate. This affinity chromatography, however, is not useful for purifying AAV4 and AAV5, because these serotypes lack heparin-binding activity. We have developed simple ion exchange high-performance liquid chromatography (HPLC) method for purifying different AAV serotypes that does not rely on the affinity of the viruses for heparin. The protocol is fast, efficient, and yields highly infectious material. Analysis of the highly purified virus indicated that more than 90% of the particles contained genomes and were more active than virus purified by cesium chloride (CsCl) gradient purification. This procedure is scalable and can easily be streamlined for large-scale production of recombinant adeno-associated virus (rAAV), regardless of the serotype. Ultimately, the new purification method will further the characterization of rAAV of different serotypes as vectors for gene therapy applications.