Engineering Allosteric Regulation into Biological Catalysts

Enzymes and ribozymes constitute two classes of biological catalysts. The activity of many natural enzymes is regulated by the binding of ligands that have different structures than their substrates; these ligands are consequently called allosteric effectors. In most allosteric enzymes, the allosteric binding site lies far away from the active site. This implies that communication pathways must exist between these sites. While mechanisms of allosteric regulation were developed more than forty years ago, they continue to be revisited regularly. The improved understanding of these mechanisms has led in the past two decades to projects to transform several unregulated enzymes into allosterically regulated ones either by rational design or directed evolution techniques. More recently, ribozymes have also been the object of similar successful engineering efforts. In this review, after briefly summarising recent progress in the theories of allosteric regulation, several strategies to engineer allosteric regulations in enzymes and ribozymes are described and compared. These redesigned biological catalysts find applications in a variety of areas.     

Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators

The physiological function of free fatty acids (FFAs) has long been regarded as indirect in terms of their activities as educts and products in metabolic pathways. The observation that FFAs can also act as signaling molecules at FFA receptors (FFARs), a family of G protein-coupled receptors (GPCRs), has changed the understanding of the interplay of metabolites and host responses. Free fatty acids of different chain lengths and saturation statuses activate FFARs as endogenous agonists via binding at the orthosteric receptor site. After FFAR deorphanization, researchers from the pharmaceutical industry as well as academia have identified several ligands targeting allosteric sites of FFARs with the aim of developing drugs to treat various diseases such as metabolic, (auto)inflammatory, infectious, endocrinological, cardiovascular, and renal disorders. GPCRs are the largest group of transmembrane proteins and constitute the most successful drug targets in medical history. To leverage the rich biology of this target class, the drug industry seeks alternative approaches to address GPCR signaling. Allosteric GPCR ligands are recognized as attractive modalities because of their auspicious pharmacological profiles compared to orthosteric ligands. While the majority of marketed GPCR drugs interact exclusively with the orthosteric binding site, allosteric mechanisms in GPCR biology stay medically underexploited, with only several allosteric ligands currently approved. This review summarizes the current knowledge on the biology of FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120), and GPR84, including structural aspects of FFAR1, and discusses the molecular pharmacology of FFAR allosteric ligands as well as the opportunities and challenges in research from the perspective of drug discovery.     

Discovery of Novel Allosteric Non‐Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding

Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti‐infective drug target. Currently available anti‐FPPS drugs are active‐site‐directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non‐bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure–activity relationships, and provide X‐ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none‐bone diseases.     

Proteases to Improve the Mechanical Characteristics of Durable Press Finished Cotton Fabrics

N‐Methylol reagents are conventional crosslinking agents that are still widely used in textile industry to produce crease‐resistant cotton fabrics. In this work serine proteases were used to recover the strength of fabrics, cross‐linked with N‐hydroxymethylacrylamide. Nearly one half of the strength loss of crosslinked cotton fabrics could be restored after protease treatment, while the wrinkle recovery angle (WRA) decreased only slightly. The enzymatic hydrolysis of the amide cross‐links in the durable pressed cellulose was confirmed by FT‐IR analysis and dyeability with an acid dye.

Effect of protease concentration on the tensile strength recovery, WRA and acid dye dyeability at 30 min reaction time.     

Chemical Validation of DegS As a Target for the Development of Antibiotics with a Novel Mode of Action

Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first in vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.     

Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct,Highly Flexible Allosteric Bicarbonate Binding Pocket

Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β‐methylene adenosine 5′‐triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar‐potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein.     

Discovery of a Dual SENP1 and SENP2 Inhibitor

SUMOylation is a reversible post–translational modification (PTM) involving covalent attachment of small ubiquitin-related modifier (SUMO) proteins to substrate proteins. Dysregulation of SUMOylation and deSUMOylation results in cellular malfunction and is linked to various diseases, such as cancer. Sentrin-specific proteases (SENPs) were identified for the maturation of SUMOs and the deconjugation of SUMOs from their substrate proteins. Hence, this is a promising target tackling the dysregulation of the SUMOylation process. Herein, we report the discovery of a novel protein-protein interaction (PPI) inhibitor for SENP1-SUMO1 by virtual screening and subsequent medicinal chemistry optimization of the hit molecule. The optimized inhibitor ZHAWOC8697 showed IC₅₀ values of 8.6 μM against SENP1 and 2.3 μM against SENP2. With a photo affinity probe the SENP target was validated. This novel SENP inhibitor represents a new valuable tool for the study of SUMOylation processes and the SENP-associated development of small molecule-based treatment options.     

Multi-step usage of in vivo models during rational drug design and discovery

In this article we propose a systematic development method for rational drug design while reviewing paradigms in industry, emerging techniques and technologies in the field. Although the process of drug development today has been accelerated by emergence of computational methodologies, it is a herculean challenge requiring exorbitant resources; and often fails to yield clinically viable results. The current paradigm of target based drug design is often misguided and tends to yield compounds that have poor absorption, distribution, metabolism, and excretion, toxicology (ADMET) properties. Therefore, an in vivo organism based approach allowing for a multidisciplinary inquiry into potent and selective molecules is an excellent place to begin rational drug design. We will review how organisms like the zebrafish and Caenorhabditis elegans can not only be starting points, but can be used at various steps of the drug development process from target identification to pre-clinical trial models. This systems biology based approach paired with the power of computational biology; genetics and developmental biology provide a methodological framework to avoid the pitfalls of traditional target based drug design.     

A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.     

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K_D=42 and 84 μM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K_D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.     

Synthesis and Biological Evaluation of 9‐Oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile Analogues as Potential Inhibitors of Deubiquitinating Enzymes

High‐throughput screening highlighted 9‐oxo‐9H‐indeno[1,2‐b]pyrazine‐2,3‐dicarbonitrile ( 1 ) as an active inhibitor of ubiquitin‐specific proteases (USPs), a family of hydrolytic enzymes involved in the removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1 , led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.     

Allosteric Modulators of Potential Targets Related to Alzheimer's Disease: a Review

Among neurodegenerative disorders, Alzheimer's disease (AD) is the most common type of dementia, and there is an urgent need to discover new and efficacious forms of treatment for it. Pathological patterns of AD include cholinergic dysfunction, increased β-amyloid (Aβ) peptide concentration, the appearance of neurofibrillary tangles, among others, all of which are strongly associated with specific biological targets. Interactions observed between these targets and potential drug candidates in AD most often occur by competitive mechanisms driven by orthosteric ligands that sometimes result in the production of side effects. In this context, the allosteric mechanism represents a key strategy; this can be regarded as the selective modulation of such targets by allosteric modulators in an advantageous manner, as this may decrease the likelihood of side effects. The purpose of this review is to present an overview of compounds that act as allosteric modulators of the main biological targets related to AD.     

Biochemical Screening of Potent Zika Virus Protease Inhibitors

As the Zika virus protease is an essential and well-established target for the development of antiviral agents, we biochemically screened for inhibitors using a purified recombinantly expressed form of this enzyme. As a result, we were able to identify 10 new Zika virus protease inhibitors. These compounds are natural products and showed strong inhibition in the biochemical assays. Inhibitory constants values for the compounds ranged from 5 nM to 8 μM. Among the most potent inhibitors are flavonoids like irigenol hexa-acetate (K_i=0.28 μM), katacine (K_i=0.26 μM), theaflavin gallate (K_i=0.40 μM) and hematein (K_i=0.33 μM). Inhibitors from other groups of natural products include sennoside A (K_i=0.19 μM) and gossypol (K_i=0.70 μM). Several of the obtained compounds are known for their beneficial health effects and have acceptable pharmacokinetic characteristics. Thus, they could be of interest as lead compounds for the development of important and essential Zika antiviral drugs.     

Allosteric Modulation of GPCRs of Class A by Cholesterol

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.     

Discovery of HIV Type 1 Aspartic Protease Hit Compounds through Combined Computational Approaches

A combination of computational techniques and inhibition assay experiments was employed to identify hit compounds from commercial libraries with enhanced inhibitory potency against HIV type 1 aspartic protease (HIV PR). Extensive virtual screening with the aid of reliable pharmacophore models yielded five candidate protease inhibitors. Subsequent molecular dynamics and molecular mechanics Poisson–Boltzmann surface area free‐energy calculations for the five ligand–HIV PR complexes suggested a high stability of the systems through hydrogen‐bond interactions between the ligands and the protease's flaps (Ile50/50′), as well as interactions with residues of the active site (Asp25/25′/29/29′/30/30′). Binding‐energy calculations for the three most promising compounds yielded values between ?5 and ?10 kcal mol^?1and suggested that van der Waals interactions contribute most favorably to the total energy. The predicted binding‐energy values were verified by in vitro inhibition assays, which showed promising results in the high nanomolar range. These results provide structural considerations that may guide further hit‐to‐lead optimization toward improved anti‐HIV drugs.     

Alzheimer’s Disease: Identification and Development of β‐Secretase (BACE‐1) Binding Fragments and Inhibitors by Dynamic Ligation Screening (DLS)

The application of dynamic ligation screening (DLS), a methodology for fragment‐based drug discovery (FBDD), to the aspartic protease β‐secretase (BACE‐1) is reported. For this purpose, three new fluorescence resonance energy transfer (FRET) substrates were designed and synthesized. Their kinetic parameters (V_max, K_M, and k_cat) were determined and compared with a commercial substrate. Secondly, a peptide aldehyde was designed as a chemically reactive inhibitor (CRI) based on the Swedish mutation substrate sequence. Incubation of this CRI with the protease, a FRET substrate, and one amine per well taken from an amine library, which was assembled by a maximum common substructure (MCS) approach, revealed the fragment 3‐(3‐aminophenyl)‐2H‐chromen‐2‐one ( 1 ) to be a competitive BACE‐1 inhibitor that enhanced the activity of the CRI. Irreversibly formed fragment combination products of 1 with the initial peptide sequence were active and confirmed the targeting of the active site through the ethane‐1,2‐diamine isostere. Finally, structure‐assisted combination of fragment 1 with secondary fragments that target the S1 site in hit optimization yielded novel, entirely fragment‐based BACE‐1 inhibitors with up to 30‐fold improved binding affinity. Interactions with the protein were explained by molecular modeling studies, which indicate that the new fragment combinations interact with the catalytic aspartic acid dyad, as well as with the adjacent binding sites required for potency.     

Tagged Benzoxazin-4-Ones as Novel Activity-Based Probes for Serine Proteases

Activity-based probes (ABPs) are valuable chemical tools for profiling enzymes. They have been particularly useful in the study of proteases. ABPs rely on electrophilic scaffolds that covalently modify the target enzymes. Ideally, they can be made in a fast and uncomplicated manner. Here, we explore alkyne-substituted benzoxazin-4-ones as ABPs for serine proteases, because they inhibitserine proteases covalently and their synthesis is very straightforward. We show that alkyne-tagged benzoxazin-4-ones can be used in two-step bioorthogonal tandem labeling procedures or pre-functionalized with a biotin or fluorophore. We demonstrate that these reagents can be used to label and identify various serine proteases. Therefore, we expect that tagged benzoxazin-4-ones will offer easily synthesizable tools for profiling of serine proteases.     

Activity-based probes for the study of proteases: recent advances and developments

Proteases are important targets for the treatment of human disease. Several protease inhibitors have failed in clinical trials due to a lack of in vivo specificity, indicating the need for studies of protease function and inhibition in complex, disease-related models. The tight post-translational regulation of protease activity complicates protease analysis by traditional proteomics methods. Activity-based protein profiling is a powerful technique that can resolve this issue. It uses small-molecule tools-activity-based probes-to label and analyze active enzymes in lysates, cells, and whole animals. Over the last twelve years, a wide variety of protease activity-based probes have been developed. These synthetic efforts have enabled techniques ranging from real-time in vivo imaging of protease activity to high-throughput screening of uncharacterized proteases. This Review introduces the general principles of activity-based protein profiling and describes the recent advancements in probe design and analysis techniques, which have increased the knowledge of protease biology and will aid future protease drug discovery.