Evaluating Scalable Supervised Learning for Synthesize-on-Demand Chemical Libraries.

Traditional small-molecule drug discovery is a time-consuming and costly endeavor. High-throughput chemical screening can only assess a tiny fraction of drug-like chemical space. The strong predictive power of modern machine-learning methods for virtual chemical screening enables training models on known active and inactive compounds and extrapolating to much larger chemical libraries. However, there has been limited experimental validation of these methods in practical applications on large commercially available or synthesize-on-demand chemical libraries. Through a prospective evaluation with the bacterial protein-protein interaction PriA-SSB, we demonstrate that ligand-based virtual screening can identify many active compounds in large commercial libraries. We use cross-validation to compare different types of supervised learning models and select a random forest (RF) classifier as the best model for this target. When predicting the activity of more than 8 million compounds from Aldrich Market Select, the RF substantially outperforms a naïve baseline based on chemical structure similarity. 48% of the RF's 701 selected compounds are active. The RF model easily scales to score one billion compounds from the synthesize-on-demand Enamine REAL database. We tested 68 chemically diverse top predictions from Enamine REAL and observed 31 hits (46%), including one with an IC50 value of 1.3 µM.

Discovery and Mechanism of Small Molecule Inhibitors Selective for the Chromatin-Binding Domains of Oncogenic UHRF1.

Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition.

Predicting kinase inhibitors using bioactivity matrix derived informer sets.

Prediction of compounds that are active against a desired biological target is a common step in drug discovery efforts. Virtual screening methods seek some active-enriched fraction of a library for experimental testing. Where data are too scarce to train supervised learning models for compound prioritization, initial screening must provide the necessary data. Commonly, such an initial library is selected on the basis of chemical diversity by some pseudo-random process (for example, the first few plates of a larger library) or by selecting an entire smaller library. These approaches may not produce a sufficient number or diversity of actives. An alternative approach is to select an informer set of screening compounds on the basis of chemogenomic information from previous testing of compounds against a large number of targets. We compare different ways of using chemogenomic data to choose a small informer set of compounds based on previously measured bioactivity data. We develop this Informer-Based-Ranking (IBR) approach using the Published Kinase Inhibitor Sets (PKIS) as the chemogenomic data to select the informer sets. We test the informer compounds on a target that is not part of the chemogenomic data, then predict the activity of the remaining compounds based on the experimental informer data and the chemogenomic data. Through new chemical screening experiments, we demonstrate the utility of IBR strategies in a prospective test on three kinase targets not included in the PKIS.

Practical Model Selection for Prospective Virtual Screening.

Virtual (computational) high-throughput screening provides a strategy for prioritizing compounds for experimental screens, but the choice of virtual screening algorithm depends on the data set and evaluation strategy. We consider a wide range of ligand-based machine learning and docking-based approaches for virtual screening on two protein-protein interactions, PriA-SSB and RMI-FANCM, and present a strategy for choosing which algorithm is best for prospective compound prioritization. Our workflow identifies a random forest as the best algorithm for these targets over more sophisticated neural network-based models. The top 250 predictions from our selected random forest recover 37 of the 54 active compounds from a library of 22,434 new molecules assayed on PriA-SSB. We show that virtual screening methods that perform well on public data sets and synthetic benchmarks, like multi-task neural networks, may not always translate to prospective screening performance on a specific assay of interest.

Machine Learning Consensus Scoring Improves Performance Across Targets in Structure-Based Virtual Screening.

In structure-based virtual screening, compound ranking through a consensus of scores from a variety of docking programs or scoring functions, rather than ranking by scores from a single program, provides better predictive performance and reduces target performance variability. Here we compare traditional consensus scoring methods with a novel, unsupervised gradient boosting approach. We also observed increased score variation among active ligands and developed a statistical mixture model consensus score based on combining score means and variances. To evaluate performance, we used the common performance metrics ROCAUC and EF1 on 21 benchmark targets from DUD-E. Traditional consensus methods, such as taking the mean of quantile normalized docking scores, outperformed individual docking methods and are more robust to target variation. The mixture model and gradient boosting provided further improvements over the traditional consensus methods. These methods are readily applicable to new targets in academic research and overcome the potentially poor performance of using a single docking method on a new target.

Isoform-selective inhibition of facilitative glucose transporters: elucidation of the molecular mechanism of HIV protease inhibitor binding.

Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.

Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.

Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.

Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 µM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

The natural product dihydrotanshinone I provides a prototype for uncharged inhibitors that bind specifically to the acetylcholinesterase peripheral site with nanomolar affinity.

Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.

Piperidine carbamate peptidomimetic inhibitors of the serine proteases HGFA, matriptase and hepsin.

Matriptase and hepsin are type II transmembrane serine proteases (TTSPs). Along with related S1 trypsin like serine protease HGFA (hepatocyte growth factor activator), their unregulated proteolytic activity has been associated with cancer including tumor progression and metastasis. These three proteases have two substrates in common, hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), the ligands for MET and recepteur d'origine nantais (RON) receptor tyrosine kinases. Mechanism-based tetrapeptide and benzamidine inhibitors of these proteases have been shown to block HGF/MET and MSP/RON cancer cell signaling. Herein, we have rationally designed a new class of peptidomimetic hybrid small molecule piperidine carbamate dipeptide inhibitors comparable in potency to much larger tetrapeptides. We have identified multiple compounds which have potent activity against matriptase and hepsin and with excellent selectivity over the off-target serine proteases factor Xa and thrombin.

A general strategy for diversifying complex natural products to polycyclic scaffolds with medium-sized rings.

The interrogation of complex biological pathways demands diverse small molecule tool compounds, which can often lead to important therapeutics for the treatment of human diseases. Since natural products are the most valuable source for the discovery of therapeutics, the derivatization of natural products has been extensively investigated to generate molecules for biological screenings. However, most previous approaches only modified a limited number of functional groups, which resulted in a limited number of skeleta. Here we show a general strategy for the preparation of a library of complex small molecules by combining state-of-the-art chemistry - the site-selective oxidation of C-H bonds - with reactions that expand rigid, small rings in polycyclic steroids to medium-sized rings. This library occupies a unique chemical space compared to selected diverse reference compounds. The diversification strategy developed herein for steroids can also be expanded to other types of natural products.

Identification of a novel class of RIP1/RIP3 dual inhibitors that impede cell death and inflammation in mouse abdominal aortic aneurysm models.

Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are essential mediators of cell death processes and participate in inflammatory responses. Our group recently demonstrated that gene deletion of Rip3 or pharmacological inhibition of RIP1 attenuated pathogenesis of abdominal aortic aneurysm (AAA), a life-threatening degenerative vascular disease characterized by depletion of smooth muscle cells (SMCs), inflammation, negative extracellular matrix remodeling, and progressive expansion of aorta. The goal of this study was to develop drug candidates for AAA and other disease conditions involving cell death and inflammation. We screened 1141 kinase inhibitors for their ability to block necroptosis using the RIP1 inhibitor Necrostatin-1s (Nec-1s) as a selection baseline. Positive compounds were further screened for cytotoxicity and virtual binding to RIP3. A cluster of top hits, represented by GSK2593074A (GSK'074), displayed structural similarity to the established RIP3 inhibitor GSK'843. In multiple cell types including mouse SMCs, fibroblasts (L929), bone marrow derived macrophages (BMDM), and human colon epithelial cells (HT29), GSK'074 inhibited necroptosis with an IC50 of ~3 nM. Furthermore, GSK'074, but not Nec-1s, blocked cytokine production by SMCs. Biochemical analyses identified both RIP1 and RIP3 as the biological targets of GSK'074. Unlike GSK'843 which causes profound apoptosis at high doses (>3 µM), GSK'074 showed no detectable cytotoxicity even at 20 µM. Daily intraperitoneal injection of GSK'074 at 0.93 mg/kg significantly attenuated aortic expansion in two mouse models of AAA (calcium phosphate: DMSO 66.06 ± 9.17% vs GSK'074 27.36 ± 8.25%, P < 0.05; Angiotensin II: DMSO 85.39 ± 15.76% vs GSK'074 36.28 ± 5.76%, P < 0.05). Histologically, GSK'074 treatment diminished cell death and macrophage infiltration in aneurysm-prone aortae. Together, our data suggest that GSK'074 represents a new class of necroptosis inhibitors with dual targeting ability to both RIP1 and RIP3. The high potency and minimum cytotoxicity make GSK'074 a desirable drug candidate of pharmacological therapies to attenuate AAA progression and other necroptosis related diseases.

Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics.

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.

Identification of 4-Amino-Thieno[2,3-d]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis.

Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of M. tuberculosis resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of M. tuberculosis To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-d]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3-d]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc1 inhibitors. Therefore, 4-amino-thieno[2,3-d]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome bc1 complex.IMPORTANCE The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in Mycobacterium tuberculosis, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome bc1 has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit M. tuberculosis growth in vitro These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-d]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.

A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein-Protein Interactions in an Academic Screening Facility.

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein-protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration-response curves with IC50 values of <40 µM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

Small Molecule Inhibitors of Metabolic Enzymes Repurposed as a New Class of Anthelmintics.

The enormous prevalence of infections caused by parasitic nematodes worldwide, coupled to the rapid emergence of their resistance to commonly used anthelmintic drugs, presents an urgent need for the discovery of new drugs. Herein, we have identified several classes of small molecules with broad spectrum activity against these pathogens. Previously, we reported the identification of carnitine palmitoyltransferases (CPTs) as a representative class of enzymes as potential targets for metabolic chokepoint intervention that was elucidated from a combination of chemogenomic screening and experimental testing in nematodes. Expanding on these previous findings, we have discovered that several chemical classes of known small molecule inhibitors of mammalian CPTs have potent activity as anthelmintics. Cross-clade efficacy against a broad spectrum of adult parasitic nematodes was demonstrated for multiple compounds from different series. Several analogs of these initial hit compounds were designed and synthesized. The compounds we report represent a good starting point for further lead identification and optimization for development of new anthelmintic drugs with broad spectrum activity and a novel mechanism of action.

Characterization of parasite-specific indels and their proposed relevance for selective anthelminthic drug targeting.

Insertions and deletions (indels) are important sequence variants that are considered as phylogenetic markers that reflect evolutionary adaptations in different species. In an effort to systematically study indels specific to the phylum Nematoda and their structural impact on the proteins bearing them, we examined over 340,000 polypeptides from 21 nematode species spanning the phylum, compared them to non-nematodes and identified indels unique to nematode proteins in more than 3000 protein families. Examination of the amino acid composition revealed uneven usage of amino acids for insertions and deletions. The amino acid composition and cost, along with the secondary structure constitution of the indels, were analyzed in the context of their biological pathway associations. Species-specific indels could enable indel-based targeting for drug design in pathogens/parasites. Therefore, we screened the spatial locations of the indels in the parasite's protein 3D structures, determined the location of the indel and identified potential unique drug targeting sites. These indels could be confirmed by RNA-Seq data. Examples are presented illustrating the close proximity of some indels to established small-molecule binding pockets that can potentially facilitate selective targeting to the parasites and bypassing their host, thus reducing or eliminating the toxicity of the potential drugs. This study presents an approach for understanding the adaptation of pathogens/parasites at a molecular level, and outlines a strategy to identify such nematode-selective targets that remain essential to the organism. With further experimental characterization and validation, it opens a possible channel for the development of novel treatments with high target specificity, addressing both host toxicity and resistance concerns.

α-Ketobenzothiazole Serine Protease Inhibitors of Aberrant HGF/c-MET and MSP/RON Kinase Pathway Signaling in Cancer.

Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types.

Hopeahainol A binds reversibly at the acetylcholinesterase (AChE) peripheral site and inhibits enzyme activity with a novel higher order concentration dependence.

Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer's disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of Hopea hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 µM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.

Tumor-Specific Binding of Radiolabeled PEGylated GIRLRG Peptide: A Novel Agent for Targeting Cancers.

Cancer-specific targeting sparing normal tissues would significantly enhance cancer therapy outcomes and reduce cancer-related mortality. One approach is to target receptors or molecules that are specifically expressed on cancer cells. Peptides as cancer-specific targeting agents offer advantages such as ease of synthesis, low antigenicity, and enhanced diffusion into tissues. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum stress chaperone that regulates the unfolded protein response and is overexpressed in various cancers. In this study, we evaluated GIRLRG peptide that specifically targets GRP78 for cancer-specific binding (in vitro) and noninvasive tumor imaging (in vivo). METHODS: GIRLRG peptide was modeled into the GRP78 ATPase domain using computational modeling. Surface plasmon resonance studies were performed to determine the affinity of GIRLRG peptide to GRP78 protein. GIRLRG was conjugated with PEG to prolong its circulation in mice. Tumor binding efficacy of PEG-GIRLRG peptide was evaluated in nude mice bearing heterotopic cervical (HT3), esophageal (OE33), pancreatic (BXPC3), lung (A549), and glioma (D54) tumors. Nano-SPECT/CT imaging of the mice was performed 48 and 72 h after injection with 111In-labeled PEG-GIRLRG or PEG-control peptide. Post-SPECT biodistribution studies were performed 96 h after injection of the radiolabeled peptides. RESULTS: Using molecular modeling and surface plasmon resonance, we identified that GIRLRG was binding with an affinity constant of 2.16 × 10-3 M in the ATPase domain of GRP78. GIRLRG peptide specifically bound to cervical, lung, esophageal, and glioma cells. SPECT imaging revealed that 111In-PEG-GIRLRG specifically bound to cervical, esophageal, pancreatic, lung, and brain tumors. Post-SPECT biodistribution data also validated the SPECT imaging results. CONCLUSION: GIRLRG peptide specifically binds to the ATPase domain of GRP78. Radiolabeled PEG-GIRLRG could be used to target various cancers. Further studies would be required to translate PEG-GIRLRG peptide into the clinic.