Shraddha Thakkar

DILIrank: the largest reference drug list ranked by the risk for developing drug-induced liver injury in humans.

This paper provides the largest, revised drug reference list annotated and ranked by the risk for developing hepatotoxicity in humans (DILIrank). We created the new DILIrank list by complementing the previously used drug-labeling information together with existing evidence of clinical causality assessments. High-throughput methods are powerful tools to develop predictive models for assessing drug-induced liver injury (DILI). However, the development of predictive models requires a drug reference list with an accurate annotation of DILI risk in humans. We previously developed a DILI annotation schema based on information curated from the US Food and Drug Administration (FDA)-approved drug labeling for 287 drugs. In this article, we refine the schema by weighing the evidence of causality (i.e., a verification process to evaluate a drug as the cause of DILI) and generate a data set that ranks the DILI risk (DILIrank) in humans for 1036 FDA-approved drugs, providing the largest annotated data set of such drugs in the public domain.Copyright

Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering

Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP.

Structure-based Design Targeted at LOX-1, a Receptor for Oxidized Low-Density Lipoprotein

Atherosclerosis related cardiovascular diseases continue to be the primary cause of mortality in developed countries. The elevated level of low density lipoprotein (LDL) is generally considered to be the driver of atherosclerosis, but recent years have seen a shift in this perception in that the vascular plaque buildup is mainly caused by oxidized LDL (ox-LDL) rather than native-LDL. The scavenger receptor LOX-1 found in endothelial cells binds and internalizes ox-LDL which leads to the initiation of plaque formation in arteries. Using virtual screening techniques, we identified a few potential small molecule inhibitors of LOX-1 and tested their inhibitory potential using differential scanning fluorimetry and various cellular assays. Two of these molecules significantly reduced the uptake of ox-LDL by human endothelial cells, LOX-1 transcription and the activation of ERK1/2 and p38 MAPKs in human endothelial cells. In addition, these molecules suppressed ox-LDL-induced VCAM-1 expression and monocyte adhesion onto human endothelial cells demonstrating their therapeutic potential.

Molecular Dynamics Guided Design of Tocoflexol: A New Radioprotectant Tocotrienol with Enhanced Bioavailability

Preclinical Research

A Nanotechnology-Based Platform for Extending the Pharmacokinetic and Binding Properties of Anti-methamphetamine Antibody Fragments

To address the need for effective medications to aid in the treatment of methamphetamine (METH) abuse, we used a nanotechnology approach to customize the in vivo behavior of an anti-METH single chain antibody (scFv7F9Cys). Anti-METH scFv7F9Cys was conjugated to dendrimer nanoparticles via a polyethylene glycol (PEG) linker to generate high-order conjugates termed dendribodies. We found that the high affinity (KD = 6.2 nM) and specificity for METH was unchanged after nanoparticle conjugation. The dendribodies were administered in an i.v. bolus to male Sprague Dawley rats after starting a s.c. infusion of METH. The PCKN values for clearance and volume of distribution of scFv7F9Cys after conjugation to dendrimers decreased 45 and 1.6-fold respectively, and the terminal elimination half-life increased 20-fold. Organ distribution of scFv7F9Cys and dendribody in blood and urine agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both experimental medications. We have thus successfully developed a novel multivalent METH-binding nanomedicine by conjugating multiple anti-METH scFvs to dendrimer nanoparticles, extending the scFv half-life from 1.3 (±0.3) to 26 (±2.6) hr. These data suggest that the dendribody design could be a feasible platform for generating multivalent antibodies with customizable PCKN profiles.

Heteroaromatic analogs of the resveratrol analog DMU-212 as potent anti-cancer agents.

Heteroaromatic analogs of DMU-212 (8-15) have been synthesized and evaluated for their anti-cancer activity against a panel of 60 human cancer cell lines. These novel analogs contain a trans-3,4,5-trimethoxystyryl moiety attached to the C2 position of indole, benzofuran, benzothiazole or benzothiophene ring (8, 11, 13 and 14, respectively) and showed potent growth inhibition in 85% of the cancer cell lines examined, with GI50 values <1 μM. Interestingly, trans-3,4- and trans-3,5-dimethoxystyryl DMU-212 analogs 9, 10, 12 and 15 exhibited significantly less growth inhibition than their 3,4,5-trimethoxystyryl counterparts, suggesting that the trans-3,4,5-trimethoxystyryl moiety is an essential structural element for the potent anti-cancer activity of these heterocyclic DMU-212 analogs. Molecular modeling studies showed that the four most active compounds (8, 11, 13 and 14) all bind to the colchicine binding site on tubulin, and that their binding modes are similar to that of DMU-212.

Development of Decision Forest Models for Prediction of Drug-Induced Liver Injury in Humans Using A Large Set of FDA-approved Drugs

Drug-induced liver injury (DILI) presents a significant challenge to drug development and regulatory science. The FDA’s Liver Toxicity Knowledge Base (LTKB) evaluated >1000 drugs for their likelihood of causing DILI in humans, of which >700 drugs were classified into three categories (most-DILI, less-DILI, and no-DILI). Based on this dataset, we developed and compared 2-class and 3-class DILI prediction models using the machine learning algorithm of Decision Forest (DF) with Mold2 structural descriptors. The models were evaluated through 1000 iterations of 5-fold cross-validations, 1000 bootstrapping validations and 1000 permutation tests (that assessed the chance correlation). Furthermore, prediction confidence analysis was conducted, which provides an additional parameter for proper interpretation of prediction results. We revealed that the 3-class model not only had a higher resolution to estimate DILI risk but also showed an improved capability to differentiate most-DILI drugs from no-DILI drugs in comparison with the 2-class DILI model. We demonstrated the utility of the models for drug ingredients with warnings very recently issued by the FDA. Moreover, we identified informative molecular features important for assessing DILI risk. Our results suggested that the 3-class model presents a better option than the binary model (which most publications are focused on) for drug safety evaluation.

Succinamide derivatives of melampomagnolide B and their anti-cancer activities

A series of succinamide derivatives of melampomagnolide B have been synthesized by coupling MMB monosuccinate (2) with various heterocyclic amines to afford compounds 3a-3l. MMB monosuccinate was also reacted with terminal diaminoalkanes to afford dimeric succinamido analogs of MMB (4a-4h). These succinamide analogs of MMB were evaluated for their anti-cancer activity against a panel of sixty human cancer cell lines. Analogs 3d-3i and dimers 4f-4g exhibited promising anti-cancer activity with GI50 values ranging from 0.28 to 33.5µM against most of the cell lines in the panel. The dimeric analogs 4f and 4g were identified as lead compounds with GI50 values in the nanomolar range (GI50=280-980nM) against several cell lines in the panel; i.e. leukemia cell lines CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226 and SR; and solid tumor cell lines NCI-H522 (non-small cell lung cancer), SW-620 and HCT-116 (colon cancer), LOX IMVI (melanoma), RXF 393 (renal cancer), and MCF7, BT-549 and MDA-MB-468 (breast cancer). Succinamide analogs 3a, 3c-3l and 4b-4h were also evaluated for their apoptotic activity against M9-ENL1 acute myelogenous leukemia cells; compounds 3h-3j and 4g were equipotent with parthenolide, exhibiting LC50 values in the range 4.1-8.1μM. Molecular docking studies indicate that these molecules interact covalently with the highly conserved Cys-46 residue of the N-terminal lobe (1-109) of human IKKβ to inhibit the NFκB transcription factor complex, resulting in down-regulation of anti-apoptotic genes under NFκB control.

175 Structure-based engineering to generate high-affinity immunotherapy for the drug of abuse

Methamphetamine (METH) abuse is a major threat in the USA and worldwide without any FDA approved medications. Anti-METH antibody antagonists block or slow the rate of METH entry into the brain and have shown efficacy in preclinical studies (Peterson, Laurenzana, Atchley, Hendrickson, & Owens, 2008). A key determinant of the antibody’s efficacy is its affinity for METH and we attempted to enhance the efficacy by designing mutations to alter the shape or the electrostatic character of the binding pocket. Towards this goal, we developed a single chain anti-METH antibody fragment (scFv6H4) from a parent IgG (1). The crystal structure of scFv-6H4 in complex with METH was determined (Celikel, Peterson, Owens, & Varughese, 2009). Based on its elucidated binding interactions, we designed point mutations in the binding pocket to improve its affinity for METH and amphetamine (AMP), the active metabolite of METH. The mutants, scFv-S93T,-I37 M and -Y34 M were cloned, expressed in yeast and tested for affinity against METH and AMP. Two mutants showed enhanced binding affinity for METH: scFv-I37 M by 1.3-fold and scFv-S93T by 2.6-fold. Additionally, all the mutants showed increase in affinity for AMP: scFv-I37 M by 56-fold, scFv-S93T by 17-fold and scFvY34 M by 5-fold. Crystal structure for one of the high-affinity mutant, scFv-S93T, in complex with METH was determined (Figure 1). Binding pocket of the mutant is more hydrophobic in comparison with the wild type. ScFv-6H4 binds METH in a deep pocket containing two water molecules. The substitution of a serine residue by a threonine leads to the expulsion of a water molecule (Figure 2), relieving some unfavorable contacts between the hydrocarbon atoms of METH and the water molecule and increasing the affinity to sub-nanomolar range. Therefore, the present study shows that efficacy could be enhanced by altering the hydrophobicity or the shape of the binding pocket.

Antioxidant Tocols as Radiation Countermeasures (Challenges to be Addressed to Use Tocols as Radiation Countermeasures in Humans)

Radiation countermeasures fall under three categories, radiation protectors, radiation mitigators, and radiation therapeutics. Radiation protectors are agents that are administered before radiation exposure to protect from radiation-induced injuries by numerous mechanisms, including scavenging free radicals that are generated by initial radiochemical events. Radiation mitigators are agents that are administered after the exposure of radiation but before the onset of symptoms by accelerating the recovery and repair from radiation-induced injuries. Whereas radiation therapeutic agents administered after the onset of symptoms act by regenerating the tissues that are injured by radiation. Vitamin E is an antioxidant that neutralizes free radicals generated by radiation exposure by donating H atoms. The vitamin E family consists of eight different vitamers, including four tocopherols and four tocotrienols. Though alpha-tocopherol was extensively studied in the past, tocotrienols have recently gained attention as radiation countermeasures. Despite several studies performed on tocotrienols, there is no clear evidence on the factors that are responsible for their superior radiation protection properties over tocopherols. Their absorption and bioavailability are also not well understood. In this review, we discuss tocopherol’s and tocotrienol’s efficacy as radiation countermeasures and identify the challenges to be addressed to develop them into radiation countermeasures for human use in the event of radiological emergencies.