A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation.

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. RESULTS: Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23-79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99%. CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

Exploiting a water network to achieve enthalpy-driven, bromodomain-selective BET inhibitors.

Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC50 values comparable to BET inhibitor (BETi) clinical candidates.

Ligand binding mode prediction by docking: mdm2/mdmx inhibitors as a case study.

The p53-binding domains of Mdm2 and Mdmx, two negative regulators of the tumor suppressor p53, are validated targets for cancer therapeutics, but correct binding poses of some proven inhibitors, particularly the nutlins, have been difficult to obtain with standard docking procedures. Virtual screening pipelines typically draw from a database of compounds represented with 1D or 2D structural information from which one or more 3D conformations must be generated. These conformations are then passed to a docking algorithm that searches for optimal binding poses on the target protein. This work tests alternative pipelines using several commonly used conformation generation programs (LigPrep, ConfGen, MacroModel, and Corina/Rotate) and docking programs (GOLD, Glide, MOE-dock, and AutoDock Vina) for their ability to reproduce known poses for a series of Mdmx and/or Mdm2 inhibitors, including several nutlins. Most combinations of these programs using default settings fail to find correct poses for the nutlins but succeed for all other compounds. Docking success for the nutlin class requires either computationally intensive conformational exploration or an "anchoring" procedure that incorporates knowledge of the orientation of the central imidazoline ring.

Macro histone H2A1.2 (macroH2A1) protein suppresses mitotic kinase VRK1 during interphase.

VRK1-mediated phosphorylation of histone H3 should be restricted in mitosis for consistent cell cycling, and defects in this process trigger cellular catastrophe. However, an interphasic regulator against VRK1 has not been actually investigated so far. Here, we show that the histone variant macrodomain-containing histone H2A1.2 functions as a suppressor against VRK1 during interphase. The level of macroH2A1.2 was markedly reduced in the mitotic phase, and the macroH2A1.2-mediated inhibition of histone H3 phosphorylation occurred mainly during interphase. We also found direct interaction and binding features between VRK1 and macroH2A1.2 by NMR spectroscopy. Hence, our findings might provide valuable insight into the underlying molecular mechanism regarding an epigenetic regulation of histone H3 during the cell cycle.

Identification of key amino acid residues in OqxB mediated efflux of fluoroquinolones using site-directed mutagenesis.

OqxB belongs to the RND (Resistance-Nodulation-Division) efflux pump family, recognized widely as a major contributor towards enhancing antimicrobial resistance. It is known to be predominantly present in all Klebsiella spp. and is attributed for its role in increasing resistance against an array of antibiotics like nitrofurantoin, quinolones, ß-lactams and colistin. However, the presence of oqxB encoding this efflux pump is not limited only to Klebsiella spp., but is also found to occur via horizontal gene transfer in other bacterial genera like Escherichia coli, Enterobacter cloacae and Salmonella spp. Recently, we reported the crystal structure of OqxB and its structure-function relationship required for the efflux of fluoroquinolones. Extending these findings further, we characterized the structural architecture of this efflux pump along with identifying some critical amino acids at the substrate binding domain of OqxB. Based on our in silico modelling studies, both hydrophobic residues (F180, L280, L621, F626) and polar residues (R48, E50, E184, R157, R774) were found to be located at this site. The present work reports the importance of these key amino acid residues and the crucial ion-pair interactions at the substrate-binding pocket, thereby establishing their role in OqxB mediated efflux and the resultant resistance development against fluoroquinolones.

NMR solution structure of human vaccinia-related kinase 1 (VRK1) reveals the C-terminal tail essential for its structural stability and autocatalytic activity.

Vaccinia-related kinase 1 (VRK1) is one of the mitotic kinases that play important roles in cell cycle, nuclear condensation, and transcription regulation. Kinase domain structures of two other VRK family members (VRK2 and VRK3) have been determined previously. However, the structure of VRK1, the most extensively studied and constitutively active VRK member, is yet to be characterized. Here, we present the nuclear magnetic resonance (NMR) solution structure of a catalytically active form of human VRK1 with its extended C-terminal tail (residues 1-361). The NMR structure of human VRK1 reveals that the C-terminal tail orients toward the catalytic site and forms a number of interactions that are critical for structural stability and catalysis. The role of this unique C-terminal tail was further investigated by deletion mutant studies where deletion of the terminal tail resulted in a dramatic reduction in the autocatalytic activity of VRK1. NMR titration studies carried out with ATP or an ATP analog confirm that ATP/ATP analogs interact with all of the crucial residues present in important motifs of the protein kinase such as the hinge region, catalytic loop, DYG motif, and thereby suggest that the catalytic domain of VRK1 is not atypical. In addition to the conventional interactions, some of the residues present on the extended C-terminal tail also interact with the ligands. These observations also substantiate the role of the extended C-terminal tail in the biological activity of VRK1.

Role of Cel5H protein surface amino acids in binding with clay minerals and measurements of its forces.

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae.

OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-ß-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.

Azaindole Based Potentiator of Antibiotics against Gram-Negative Bacteria.

We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane's integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of ß-lactam antibiotics and ß-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While ß-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer.

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Binding mode analyses and pharmacophore model development for sulfonamide chalcone derivatives, a new class of alpha-glucosidase inhibitors.

Sulfonamide chalcone derivatives are a new class of non-saccharide compounds that effectively inhibit glucosidases which are the major targets in the treatment of Type 2 diabetes and HIV infection. Our aim is to explore their binding mode of interaction at the active site by comparing with the sugar derivatives and to develop a pharmacophore model which would represent the critical features responsible for alpha-glucosidase inhibitory activity. The homology modeled structure of Saccharomyces cerevisiae alpha-glucosidase was built and used for molecular docking of non-sugar/sugar derivatives. The validated docking results projected the crucial role of NH group in the binding of sugar/non-sugar derivatives to the active site. Ligplot analyses revealed that Tyr71, and Phe177 form hydrophobic interactions with sugar/non-sugar derivatives by holding the terminal glycosidic ring mimics. Molecular dynamic (MD) simulation studies were performed for protein alone and with chalcone derivative to prove its binding mechanism as shown by docking/Ligplot results. It would also help to substantiate the homology modeled structure stability. With the knowledge of the crucial interactions between ligand and protein from docking and MD simulation studies, features for pharmacophore model development were chosen. The CATALYST/HipHop was used to generate a five featured pharmacophore model with a training set of five non-sugar derivatives. As validation, all the crucial features of the model were perfectly mapped onto the 3D structures of the sugar derivatives as well as the newly tested non-sugar derivatives. Thus, it can be useful in virtual screening for finding new non-sugar derivatives as alpha-glucosidase inhibitors.

The role of ZA channel water-mediated interactions in the design of bromodomain-selective BET inhibitors.

The Bromodomain and Extra-Terminal domain (BET) family of proteins are involved in the regulation of gene transcription, and their dysregulation is implicated in several diseases including cancer. BET proteins contain two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine residues and appear to have distinct biological roles. We compared several published co-crystal structures and found five positions near the substrate binding pocket that vary between BET bromodomains. One position located in the ZA loop has unique properties. In BRD2-4, this residue is glutamine in BD1 and lysine in BD2; in BRDT, this residue is arginine in BD1 and asparagine in BD2. Using molecular modeling, we identified differences in the water-mediated network at this position between bromodomains. Molecular dynamics simulations helped rationalize the observed bromodomain selectivity for exemplar BET inhibitors and a congeneric series of tetrahydroquinolines (THQ) that differed by a single heteroatom near the ZA channel. The 2-furan SJ830599, the most BD2-selective THQ analog, did not disrupt the water-mediated networks in either domain, but was electrostatically-repulsed by the specific arrangement of the W5 water dipole in BD1. Our work underscores the value of exploring water-mediated interactions to study ligand binding, and highlights the difficulty of optimizing polar interactions due to high desolvation penalties. Finally, we suggest further modifications to THQ-based BET inhibitors that would increase BD2-selectivity in BRD2-4, while minimizing affinity for one or both bromodomains of BRDT.

Correction: Suprafenacine, an Indazole-Hydrazide Agent, Targets Cancer Cells Through Microtubule Destabilization.

[This corrects the article DOI: 10.1371/journal.pone.0110955.].

Nitrothiophene carboxamides, a novel narrow spectrum antibacterial series: Mechanism of action and Efficacy.

The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.

Pharmacophore identification and virtual screening for methionyl-tRNA synthetase inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes involved in protein biosynthesis in all living organisms and are an unexploited antibacterial targets, as many strains of bacteria have become resistant to all established classes of antibiotics. Therefore, the main aim of this study is to discover new lead molecules which would be useful as anti-bacterial compounds. Pharmacophore models were developed by using CATALYST HypoGen with a training set of 29 diverse methionyl-tRNA synthetase (MetRS) inhibitors. The best quantitative pharmacophore hypothesis (Hypo1) obtained a correlation coefficient of 0.975, root mean square deviation (RMSD) of 0.55 and cost difference (null cost-total cost) of 70.32. This Hypo1 was validated by two methods, first by using 104 test set molecules which resulted a correlation of 0.926 between HypoGen estimated activities versus experimental activities and secondly by Cat-Scramble validation method. This validated pharmacophore model was further used for screening databases for discovery of new MetRS inhibitors. The new lead compounds were further analyzed for drug-like properties. Homology modeled structure of Staphylococcus aureus MetRS was built and molecular docking studies were performed with many inhibitors using the newly built protein structure. Finally, it was found that the new leads exhibited good estimated inhibitory activity, calculated binding properties similar to experimentally proven compounds and also favorable drug-like properties.

Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors.

A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii.

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

Performance of a docking/molecular dynamics protocol for virtual screening of nutlin-class inhibitors of Mdmx.

A virtual screening protocol involving docking and molecular dynamics has been tested against the results of fluorescence polarization assays testing the potency of a series of compounds of the nutlin class for inhibition of the interaction between p53 and Mdmx, an interaction identified as a driver of certain cancers. The protocol uses a standard docking method (AutoDock) with a cutoff based on the AutoDock score (ADscore), followed by molecular dynamics simulation with a cutoff based on root-mean-square-deviation (RMSD) from the docked pose. An analysis of the experimental and computational results shows modest performance of ADscore alone, but dramatically improved performance when RMSD is also used.

Monitoring Ligand-Induced Protein Ordering in Drug Discovery.

While the gene for p53 is mutated in many human cancers causing loss of function, many others maintain a wild-type gene but exhibit reduced p53 tumor suppressor activity through overexpression of the negative regulators, Mdm2 and/or MdmX. For the latter mechanism of loss of function, the activity of endogenous p53 can be restored through inhibition of Mdm2 or MdmX with small molecules. We previously reported a series of compounds based upon the Nutlin-3 chemical scaffold that bind to both MdmX and Mdm2 [Vara, B. A. et al. (2014) Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: A platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition. J. Org. Chem. 79, 6913-6938]. Here we present the first solution structures based on data from NMR spectroscopy for MdmX in complex with four of these compounds and compare them with the MdmX:p53 complex. A p53-derived peptide binds with high affinity (Kd value of 150nM) and causes the formation of an extensive network of hydrogen bonds within MdmX; this constitutes the induction of order within MdmX through ligand binding. In contrast, the compounds bind more weakly (Kd values from 600nM to 12µM) and induce an incomplete hydrogen bond network within MdmX. Despite relatively weak binding, the four compounds activated p53 and induced p21(Cip1) expression in retinoblastoma cell lines that overexpress MdmX, suggesting that they specifically target MdmX and/or Mdm2. Our results document structure-activity relationships for lead-like small molecules targeting MdmX and suggest a strategy for their further optimization in the future by using NMR spectroscopy to monitor small-molecule-induced protein order as manifested through hydrogen bond formation.