Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Not All Binding Sites Are Equal: Site Determination and Folding State Analysis of Gas-Phase Protein-Metallodrug Adducts.

Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.

ß-Lactoglobulin variants as potential carriers of pramoxine: Comprehensive structural and biophysical studies.

ß-Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to ß-lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography were used to understand the mechanisms of BLG-PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80-fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the ß-barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.

Discovering Biomarkers and Pathways Shared by Alzheimer's Disease and Ischemic Stroke to Identify Novel Therapeutic Targets.

Background and objectives: Alzheimer's disease (AD) is a progressive neurodegenerative disease that results in severe dementia. Having ischemic strokes (IS) is one of the risk factors of the AD, but the molecular mechanisms that underlie IS and AD are not well understood. We thus aimed to identify common molecular biomarkers and pathways in IS and AD that can help predict the progression of these diseases and provide clues to important pathological mechanisms. Materials and Methods: We have analyzed the microarray gene expression datasets of IS and AD. To obtain robust results, combinatorial statistical methods were used to analyze the datasets and 26 transcripts (22 unique genes) were identified that were abnormally expressed in both IS and AD. Results: Gene Ontology (GO) and KEGG pathway analyses indicated that these 26 common dysregulated genes identified several altered molecular pathways: Alcoholism, MAPK signaling, glycine metabolism, serine metabolism, and threonine metabolism. Further protein-protein interactions (PPI) analysis revealed pathway hub proteins PDE9A, GNAO1, DUSP16, NTRK2, PGAM2, MAG, and TXLNA. Transcriptional and post-transcriptional components were then identified, and significant transcription factors (SPIB, SMAD3, and SOX2) found. Conclusions: Protein-drug interaction analysis revealed PDE9A has interaction with drugs caffeine, γ-glutamyl glycine, and 3-isobutyl-1-methyl-7H-xanthine. Thus, we identified novel putative links between pathological processes in IS and AD at transcripts levels, and identified possible mechanistic and gene expression links between IS and AD.

The drug diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1.

The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.

Transported substrate determines exchange rate in the multidrug resistance transporter EmrE.

EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized by this transporter. We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Here we show that even within this limited series, the rate of interconversion between the inward- and outward-facing states of EmrE varies over 3 orders of magnitude. Thus, the identity of the bound substrate controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and is correlated with substrate hydrophobicity within the tetrahedral substrate series. Substrate identity influences both the ground-state and transition-state energies for the conformational exchange process, highlighting the coupling between substrate binding and transport required for alternating access antiport.

Oxicams bind in a novel mode to the cyclooxygenase active site via a two-water-mediated H-bonding Network.

Oxicams are widely used nonsteroidal anti-inflammatory drugs (NSAIDs), but little is known about the molecular basis of the interaction with their target enzymes, the cyclooxygenases (COX). Isoxicam is a nonselective inhibitor of COX-1 and COX-2 whereas meloxicam displays some selectivity for COX-2. Here we report crystal complexes of COX-2 with isoxicam and meloxicam at 2.0 and 2.45 angstroms, respectively, and a crystal complex of COX-1 with meloxicam at 2.4 angstroms. These structures reveal that the oxicams bind to the active site of COX-2 using a binding pose not seen with other NSAIDs through two highly coordinated water molecules. The 4-hydroxyl group on the thiazine ring partners with Ser-530 via hydrogen bonding, and the heteroatom of the carboxamide ring of the oxicam scaffold interacts with Tyr-385 and Ser-530 through a highly coordinated water molecule. The nitrogen atom of the thiazine and the oxygen atom of the carboxamide bind to Arg-120 and Tyr-355 via another highly ordered water molecule. The rotation of Leu-531 in the structure opens a novel binding pocket, which is not utilized for the binding of other NSAIDs. In addition, a detailed study of meloxicam·COX-2 interactions revealed that mutation of Val-434 to Ile significantly reduces inhibition by meloxicam due to subtle changes around Phe-518, giving rise to the preferential inhibition of COX-2 over COX-1.

The DrrAB efflux system of Streptomyces peucetius is a multidrug transporter of broad substrate specificity.

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics, doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon, which codes for a dedicated ABC (ATP binding cassette)-type transporter for the export of these two closely related antibiotics. Because of its dedicated nature, the DrrAB system is believed to belong to the category of single-drug transporters. However, whether it also contains specificity for other known substrates of multidrug transporters has never been tested. In this study we demonstrate under both in vivo and in vitro conditions that the DrrAB system can transport not only doxorubicin but is also able to export two most commonly studied MDR substrates, Hoechst 33342 and ethidium bromide. Moreover, we demonstrate that many other substrates (including verapamil, vinblastine, and rifampicin) of the well studied multidrug transporters inhibit DrrAB-mediated Dox transport with high efficiency, indicating that they are also substrates of the DrrAB pump. Kinetic studies show that inhibition of doxorubicin transport by Hoechst 33342 and rifampicin occurs by a competitive mechanism, whereas verapamil inhibits transport by a non-competitive mechanism, thus suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. The significance of these findings in evolution of poly-specificity in drug resistance systems is discussed.

Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.

Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and ß2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.

Selective inhibition of human group IIA-secreted phospholipase A2 (hGIIA) signaling reveals arachidonic acid metabolism is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes.

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.

Novel yeast-based strategy unveils antagonist binding regions on the nuclear xenobiotic receptor PXR.

BACKGROUND: Ketoconazole binds to and antagonizes pregnane X receptor (PXR) activation. RESULTS: Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. CONCLUSION: Ketoconazole genetically interacts with specific PXR surface residues. SIGNIFICANCE: A yeast-based genetic method to discover novel nuclear receptor interactions with ligands that associate with surface binding sites is suggested. The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple "gain-of-function" mutants that were "resistant" to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications.

UV-triggered affinity capture identifies interactions between the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) and antimalarial agents in live parasitized cells.

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.

Insights into the effect of glucose on the binding between human serum albumin and the nonsteroidal anti-inflammatory drug nimesulide.

Glucose interacts with human serum albumin (HSA, the main protein responsible for the biodistribution of drugs in the bloodstream) and consequently affects the binding capacity of exogenous compounds. Thus, in this work, the interactive profile between HSA and the anti-inflammatory drug nimesulide (NMD, used mainly by patients with diabetic neuropathy to relieve acute or chronic pains) was characterized in nonglycemic, normoglycemic (80 mg/dL), and hyperglycemic (320 mg/dL) conditions by biophysics techniques. There is a spontaneous and ground-state association HSA:NMD under physiological conditions. Therefore, the Stern-Volmer constant (Ksv) can also be used to estimate the binding affinity. The Ksv values for nonglycemic, normoglycemic, and hyperglycemic conditions are around 104 M-1, indicating a moderate affinity of NMD to albumin that was slightly improved by glucose levels. Additionally, the binding is enthalpically and entropically driven mainly into subdomains IIA or IIIA. The binding perturbs weakly the α-helix content of albumin, however, glucose potentially stabilizes the tertiary structure, decreasing the structural perturbation upon NMD binding and improves the complex HSA:NMD stability. Overall, the biophysical characterization indicated that glucose levels might slightly positively impact the pharmacokinetic profile of NMD, allowing NMD to achieve its therapeutical potential without affecting drastically its effective dosages.

Unraveling the Structure of Meclizine Dihydrochloride with MicroED.

Meclizine (Antivert, Bonine) is a first-generation H1 antihistamine used in the treatment of motion sickness and vertigo. Despite its wide medical use for over 70 years, its crystal structure and the details of protein-drug interactions remained unknown. Single-crystal X-ray diffraction (SC-XRD) is previously unsuccessful for meclizine. Today, microcrystal electron diffraction (MicroED) enables the analysis of nano- or micro-sized crystals that are merely a billionth the size needed for SC-XRD directly from seemingly amorphous powder. In this study, MicroED to determine the 3D crystal structure of meclizine dihydrochloride is used. Two racemic enantiomers (R/S) are found in the unit cell, which is packed as repetitive double layers in the crystal lattice. The packing is made of multiple strong N-H-Cl- hydrogen bonding interactions and weak interactions like C-H-Cl- and pi-stacking. Molecular docking reveals the binding mechanism of meclizine to the histamine H1 receptor. A comparison of the docking complexes between histamine H1 receptor and meclizine or levocetirizine (a second-generation antihistamine) shows the conserved binding sites. This research illustrates the combined use of MicroED and molecular docking in unraveling elusive drug structures and protein-drug interactions for precision drug design and optimization.

The interplay of structure and dynamics: insights from a survey of HIV-1 reverse transcriptase crystal structures.

HIV-1 reverse transcriptase (RT) is a critical drug target for HIV treatment, and understanding the exact mechanisms of its function and inhibition would significantly accelerate the development of new anti-HIV drugs. It is well known that structure plays a critical role in protein function, but for RT, structural information has proven to be insufficient-despite enormous effort-to explain the mechanism of inhibition and drug resistance of non-nucleoside RT inhibitors. We hypothesize that the missing link is dynamics, information about the motions of the system. However, many of the techniques that give the best information about dynamics, such as solution nuclear magnetic resonance and molecular dynamics simulations, cannot be easily applied to a protein as large as RT. As an alternative, we combine elastic network modeling with simultaneous hierarchical clustering of structural and dynamic data. We present an extensive survey of the dynamics of RT bound to a variety of ligands and with a number of mutations, revealing a novel mechanism for drug resistance to non-nucleoside RT inhibitors. Hydrophobic core mutations restore active-state motion to multiple functionally significant regions of HIV-1 RT. This model arises out of a combination of structural and dynamic information, rather than exclusively from one or the other.

The effect of human serum albumin glycation on the binding of antidiabetic agent-exenatide; the multispectroscopic studies. Part II.

The glycated human serum albumin (gHSA) interaction with antidiabetic agent - exenatide (exe) was investigated by spectroscopic techniques. The fluorescence spectroscopy showed an association between exe and protein as 4.57 × 104 M-1 (290 K), 3.33 × 104 M-1 (300K) and 2.54 × 104 M-1 (310K) and the number of binding sites were 1.15, 1.09, and 1.02, respectively. The binding process occurred spontaneously and the electrostatic bonds play a predominant role in the gHSA-exe interaction. The affinity drug to gHSA was studied in the presence of metal ions (Ca2+, Cr3+, Zn2+), which are often given as a supplement to the diet. Our study indicates that metal ions have an effect on the increase of the affinity of exenatide to glycated albumin and it is the result of the metal ion-exenatide-albumin interaction. Based on CD spectroscopy it is evident that exenatide has nonsignificant effect on the protein secondary structure, but the changes are visible in the presence of metal ions. Zeta potential measurements indicate instability of the system at lower concentration of the ligand - the effect is desired in point of view of pharmacology.

Spectroscopy and dynamics of beta-lactoglobulin complexed with rifampicin.

In this paper, we report the binding interaction of milk protein, beta-lactoglobulin (BLG), with an antibiotic against tuberculosis, rifampicin (RIF). BLG intrinsic fluorescence from tryptophan (Trp) amino acids was monitored to understand protein-drug interactions. Binding parameters and stoichiometry were estimated with the help of fluorescence spectral changes. Synchronous fluorescence spectroscopy was employed to exclusively monitor the Trp and Tyrosine (Tyr) environment in the presence of RIF. With the help of steady state fluorescence at different temperatures supported by time-resolved fluorescence, we confirmed that the protein forms a static complex with RIF. Thermodynamic parameters, ΔH and ΔS values, showed the involvement of hydrophobic forces between the RIF and BLG. Competitive displacement assay with ANS confirmed the BLG calyx as the binding site for RIF. Energy transfer mechanism from Trp to RIF was attributed to the fluorescence changes in protein upon complexation. The Förster resonance energy transfer (FRET) was used to find distance, energy transfer efficiency and rate of energy transfer between donor (BLG) and acceptor (RIF). Fourier-transform infrared (FTIR) spectroscopy was utilized for estimating changes in the secondary structure of BLG induced by RIF. Molecular docking was used to visualise the binding location of RIF on BLG. Molecular dynamics (MD) simulation studies showed a consistent binding interactions between BLG and RIF during the 100 ns simulation period and this well supported the increased beta sheet content in FTIR. Overall our results establish the potential of intrinsic fluorescence of BLG in combination with biophysical tools to rationalize drug-protein interactions.Communicated by Ramaswamy H. Sarma.

Revealing new therapeutic opportunities in hypertension through network-driven integrative genetic analysis and drug target prediction approach.

Epidemiological studies have established that untreated hypertension (HTN) is a major independent risk factor for developing cardiovascular diseases (CVD), stroke, renal failure, and other conditions. Several important studies have been published to prevent and manage HTN; however, antihypertensive agents' optimal choice remains controversial. Therefore, the present study is undertaken to update our knowledge in the primary treatment of HTN, specifically in the setting of other three important diseases. MicroRNAs (miRNAs) are remarkably stable short endogenous conserved non-coding RNAs that bind to the mRNA at its (3' UTR) to regulate its gene expression by causing translational repression or mRNA degradation. Through their coordinated activities on different pathways and networks, individual miRNAs control normal and pathological cellular processes. Therefore, to identify the critical miRNA-mRNA-TF interactions, we performed systematic bioinformatics analysis. We have also employed the molecular modelling and docking approach to identify the therapeutic target that delivers novel empathies into Food and Drug Administration approved and herbal drug response physiology. Gene Expression Omnibus (GEO) was employed to identify the differentially expressed genes (DEGs) and hub genes- KNG1, HLA-DPB1, CXCL8, IL1B, and BCL2. The HTN associated feed-forward loop (FFL) network included miR-9-5p, KNG1 and AR. We employed high throughput screening to get the best interacting compounds, telmisartan and limonin, that provided a significant docking score (-13.3 and -12.0 kcal/mol) and a potential protective effect that may help to combat the impact of HTN. The present study provides novel insight into HTN etiology through the identification of mRNAs and miRNAs and associated pathways.

Crystal structure of death-associated protein kinase 1 in complex with the dietary compound resveratrol.

Death-associated protein kinase 1 (DAPK1) is a large multidomain protein with an N-terminal serine/threonine protein kinase domain. DAPK1 is considered to be a promising molecular target for the treatment of Alzheimer's disease (AD). In the present study, the inhibitory potency of resveratrol (RSV), a dietary polyphenol found in red wine, against the catalytic activity of DAPK1 was investigated. Kinetic and fluorescent probe competitive binding analyses revealed that RSV directly inhibited the catalytic activity of DAPK1 by binding to the ATP-binding site. Crystallographic analysis of DAPK1 in complex with RSV revealed that the A-ring of RSV occupied the nucleobase-binding position. Determination of the binding mode provided a structural basis for the design of more potent DAPK1 inhibitors. In conclusion, the data here clearly show that RSV is an ATP-competitive inhibitor of DAPK1, encouraging speculation that RSV may be useful for the development of AD inhibitors.

Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One Derivatives.

Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.