Future Structure-based Drug Design Research Articles

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors

α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an ‘upper’ and ‘lower’ moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure–activity relationships, and empower future structure-based drug design campaigns.

Structural and functional insights into the Pseudomonas aeruginosa glycosyltransferase WaaG and the implications for lipopolysaccharide biosynthesis

The glycosyltransferase WaaG in Pseudomonas aeruginosa (PaWaaG) is involved in synthesis of the core region of lipopolysaccharides. It is a promising target for developing adjuvants that could help in the uptake of antibiotics. Herein we have determined structures of PaWaaG in complex with the nucleotide-sugars UDP-glucose, UDP-galactose and UDP-GalNAc. Structural comparison with the homologue from Escherichia coli (EcWaaG) revealed five key differences in the sugar binding pocket. Solution-state NMR analysis showed that wildtype PaWaaG specifically hydrolyzes UDP-GalNAc and unlike EcWaaG, does not hydrolyze UDP-glucose. Furthermore, we found that a PaWaaG mutant (Y97F/T208R/N282A/T283A/T285I) designed to resemble the EcWaaG sugar binding site, only hydrolyzed UDP-glucose, underscoring the importance of the identified amino acids in substrate specificity. However, neither wildtype PaWaaG nor the PaWaaG mutant capable of hydrolyzing UDP-glucose was able to complement an E. coli ΔwaaG strain, indicating that more remains to be uncovered about the function of PaWaaG in vivo. This structural and biochemical information will guide future structure-based drug design efforts targeting PaWaaG.

A generalized protein-ligand scoring framework with balanced scoring, docking, ranking and screening powers.

Applying machine learning algorithms to protein-ligand scoring functions has aroused widespread attention in recent years due to the high predictive accuracy and affordable computational cost. Nevertheless, most machine learning-based scoring functions are only applicable to a specific task, e.g., binding affinity prediction, binding pose prediction or virtual screening, suggesting that the development of a scoring function with balanced performance in all critical tasks remains a grand challenge. To this end, we propose a novel parameterization strategy by introducing an adjustable binding affinity term that represents the correlation between the predicted outcomes and experimental data into the training of mixture density network. The resulting residue-atom distance likelihood potential not only retains the superior docking and screening power over all the other state-of-the-art approaches, but also achieves a remarkable improvement in scoring and ranking performance. We emphatically explore the impacts of several key elements on prediction accuracy as well as the task preference, and demonstrate that the performance of scoring/ranking and docking/screening tasks of a certain model could be well balanced through an appropriate manner. Overall, our study highlights the potential utility of our innovative parameterization strategy as well as the resulting scoring framework in future structure-based drug design.

Evolution of Stronger SARS-CoV-2 Variants as Revealed Through the Lens of Molecular Dynamics Simulations.

Using molecular dynamics simulations, the protein–protein interactions of the receptor-binding domain of the wild-type and seven variants of the severe acute respiratory syndrome coronavirus 2 spike protein and the peptidase domain of human angiotensin-converting enzyme 2 were investigated. These variants are alpha, beta, gamma, delta, eta, kappa, and omicron. Using 100 ns simulation data, the residue interaction networks at the protein–protein interface were identified. Also, the impact of mutations on essential protein dynamics, backbone flexibility, and interaction energy of the simulated protein–protein complexes were studied. The protein–protein interface for the wild-type, delta, and omicron variants contained several stronger interactions, while the alpha, beta, gamma, eta, and kappa variants exhibited an opposite scenario as evident from the analysis of the inter-residue interaction distances and pair-wise interaction energies. The study reveals that two distinct residue networks at the central and right contact regions forge stronger binding affinity between the protein partners. The study provides a molecular-level insight into how enhanced transmissibility and infectivity by delta and omicron variants are most likely tied to a handful of interacting residues at the binding interface, which could potentially be utilized for future antibody constructs and structure-based antiviral drug design. Supplementary InformationThe online version contains supplementary material available at 10.1007/s10930-022-10065-6.

Hybrid drug-screening strategy identifies potential SARS-CoV-2 cell-entry inhibitors targeting human transmembrane serine protease.

The spread of coronavirus infectious disease (COVID-19) is associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has risked public health more than any other infectious disease. Researchers around the globe use multiple approaches to identify an effective approved drug (drug repurposing) that treats viral infections. Most of the drug repurposing approaches target spike protein or main protease. Here we use transmembrane serine protease 2 (TMPRSS2) as a target that can prevent the virus entry into the cell by interacting with the surface receptors. By hypothesizing that the TMPRSS2 binders may help prevent the virus entry into the cell, we performed a systematic drug screening over the current approved drug database. Furthermore, we screened the Enamine REAL fragments dataset against the TMPRSS2 and presented nine potential drug-like compounds that give us clues about which kinds of groups the pocket prefers to bind, aiding future structure-based drug design for COVID-19. Also, we employ molecular dynamics simulations, binding free energy calculations, and well-tempered metadynamics to validate the obtained candidate drug and fragment list. Our results suggested three potential FDA-approved drugs against human TMPRSS2 as a target. These findings may pave the way for more drugs to be exposed to TMPRSS2, and testing the efficacy of these drugs with biochemical experiments will help improve COVID-19 treatment.Supplementary informationThe online version contains supplementary material available at 10.1007/s11224-022-01960-w.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.

Structure of the sodium-dependent phosphate transporter reveals insights into human solute carrier SLC20.

Inorganic phosphate (Pi) is a fundamental and essential element for nucleotide biosynthesis, energy supply, and cellular signaling in living organisms. Human phosphate transporter (hPiT) dysfunction causes numerous diseases, but the molecular mechanism underlying transporters remains elusive. We report the structure of the sodium-dependent phosphate transporter from Thermotoga maritima (TmPiT) in complex with sodium and phosphate (TmPiT-Na/Pi) at 2.3-angstrom resolution. We reveal that one phosphate and two sodium ions (Pi-2Na) are located at the core of TmPiT and that the third sodium ion (Nafore) is located near the inner membrane boundary. We propose an elevator-like mechanism for sodium and phosphate transport by TmPiT, with the TmPiT-Na/Pi complex adopting an inward occluded conformation. We found that disease-related hPiT variants carry mutations in the corresponding sodium- and phosphate-binding residues identified in TmPiT. Our three-dimensional structure of TmPiT provides a framework for understanding PiT dysfunction and for future structure-based drug design.

Multiscale Molecular Modeling in G Protein-Coupled Receptor (GPCR)-Ligand Studies.

G protein-coupled receptors (GPCRs) are major drug targets due to their ability to facilitate signal transduction across cell membranes, a process that is vital for many physiological functions to occur. The development of computational technology provides modern tools that permit accurate studies of the structures and properties of large chemical systems, such as enzymes and GPCRs, at the molecular level. The advent of multiscale molecular modeling permits the implementation of multiple levels of theories on a system of interest, for instance, assigning chemically relevant regions to high quantum mechanics (QM) level of theory while treating the rest of the system using classical force field (molecular mechanics (MM) potential). Multiscale QM/MM molecular modeling have far-reaching applications in the rational design of GPCR drugs/ligands by affording precise ligand binding configurations through the consideration of conformational plasticity. This enables the identification of key binding site residues that could be targeted to manipulate GPCR function. This review will focus on recent applications of multiscale QM/MM molecular simulations in GPCR studies that could boost the efficiency of future structure-based drug design (SBDD) strategies.

Multi-target, ensemble-based virtual screening yields novel allosteric KRAS inhibitors at high success rate.

RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble-based virtual screening using pocket-tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole-based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure-based drug design efforts against KRAS, a high-value target for cancer therapy.

The Growing Role of Electron Microscopy in Anti-parasitic Drug Discovery.

Parasite diseases are a huge burden on human health causing significant morbidity and mortality. However, parasite structure based drug discovery programmes have been hindered by a lack of high resolution structural information from parasite derived proteins and have often relied upon homology models from mammalian systems. The recent renaissance in electron microscopy (EM) has caused a dramatic rise in the number of structures being determined at high resolution and subsequently enabled it to be thought of as a tool in drug discovery. In this review, we discuss the challenges associated with the structural determination of parasite proteins including the difficulties in obtaining sufficient quantities of protein. We then discuss the reasons behind the resurgence in EM, how it may overcome some of these challenges and provide examples of EM derived parasite protein structures. Finally, we discuss the challenges which EM needs to overcome before it is used as a mainstream technique in anti-parasite drug discovery. This review reports the progress that has been made in obtaining sufficient quantities of proteins for structural studies and the role EM may play in future structure based drug design programs. The outlook for future structure based drug design programs against some of the most devastating parasite diseases looks promising.

The crystal structure of glucokinase from Leishmania braziliensis

Glucokinase from pathogenic protozoa of the genus Leishmania is a potential drug target for the chemotherapeutic treatment against leishmaniasis because this enzyme is located at a nodal point between two critically important metabolic pathways, glycolysis and the pentose phosphate pathway (PPP). L. braziliensis glucokinase (LbGlcK) was evaluated for its structural characterization and enzymatic performance. The enzyme catalyzes the phosphorylation of d-glucose with co-substrate ATP to yield the products G6P and ADP. LbGlcK had KM values determined as 6.61 ± 2.63 mM and 0.338 ± 0.080 mM for d-glucose and ATP, respectively. The 1.85 Å resolution X-ray crystal structure of the apo form of LbGlcK was determined and a homodimer was revealed where each subunit (both in open conformations) included the typical small and large domains. Structural comparisons were assessed in relationship to Homo sapiens hexokinase IV and Trypanosoma cruzi glucokinase. Comparisons revealed that all residues important for making hydrogen bonding interactions with d-glucose in the active site and catalysis were strictly conserved. LbGlcK was screened against four glucosamine analogue inhibitors and the stronger inhibitor of the series, HPOP-GlcN, had a Ki value of 56.9 ± 16.6 μM that exhibited competitive inhibition. For the purpose of future structure-based drug design experimentation, L. braziliensis glucokinase was observed to be very similar to T. cruzi glucokinase even though there was a 44% protein sequence identity between the two enzymes.

Molecular characterization of the interaction of sialic acid with the periplasmic binding protein from Haemophilus ducreyi

The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell. The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host-derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae SiaPs bind the sialic acid N-acetylneuraminic acid (Neu5Ac) with nanomolar affinity by forming electrostatic and hydrogen-bonding interactions. Here, we report the crystal structure of a periplasmic binding protein (SatA) of the ATP-binding cassette (ABC) transport system from the pathogenic bacterium Haemophilus ducreyi The structure of Hd-SatA in the native form and sialic acid-bound forms (with Neu5Ac and N-glycolylneuraminic acid (Neu5Gc)), determined to 2.2, 1.5, and 2.5 Å resolutions, respectively, revealed a ligand-binding site that is very different from those of the SiaPs of the TRAP transport system. A structural comparison along with thermodynamic studies suggested that similar affinities are achieved in the two classes of proteins through distinct mechanisms, one enthalpically driven and the other entropically driven. In summary, our structural and thermodynamic characterization of Hd-SatA reveals that it binds sialic acids with nanomolar affinity and that this binding is an entropically driven process. This information is important for future structure-based drug design against this pathogen and related bacteria.

Structural Basis of Splicing Modulation by Antitumor Macrolide Compounds

SF3B is a multi-protein complex essential for branch site (BS) recognition and selection during pre-mRNA splicing. Several splicing modulators with antitumor activity bind SF3B and thereby modulate splicing. Here we report the crystal structure of a human SF3B core in complex with pladienolide B (PB), a macrocyclic splicing modulator and potent inhibitor of tumor cell proliferation. PB stalls SF3B in an open conformation by acting like a wedge within a hinge, modulating SF3B's transition to the closed conformation needed to form the BS adenosine-binding pocket and stably accommodate the BS/U2 duplex. This work explains the structural basis for the splicing modulation activity of PB and related compounds, and reveals key interactions between SF3B and a common pharmacophore, providing a framework for future structure-based drug design.

Structural basis for lipid binding and mechanism of the Mycobacterium tuberculosis Rv3802 phospholipase

The Mycobacterium tuberculosis rv3802c gene encodes an essential enzyme with thioesterase and phospholipase A activity. Overexpression of Rv3802 orthologs in Mycobacterium smegmatis and Corynebacterium glutamicum increases mycolate content and decreases glycerophospholipids. Although a role in modulating the lipid composition of the unique mycomembrane has been proposed, the true biological function of Rv3802 remains uncertain. In this study, we present the first M. tuberculosis Rv3802 X-ray crystal structure, solved to 1.7 Å resolution. On the basis of the binding of PEG molecules to Rv3802, we identified its lipid-binding site and the structural basis for phosphatidyl-based substrate binding and phospholipase A activity. We found that movement of the α8-helix affords lipid binding and is required for catalytic turnover through covalent tethering. We gained insights into the mechanism of acyl hydrolysis by observing differing arrangements of PEG and water molecules within the active site. This study provides structural insights into biological function and facilitates future structure-based drug design toward Rv3802.

DXS as a target for structure-based drug design.

In this review, we analyze the enzyme DXS, the first and rate-limiting protein in the methylerythritol 4-phosphate pathway. This pathway was discovered in 1996 and is one of two known metabolic pathways for the biosynthesis of the universal building blocks for isoprenoids. It promises to offer new targets for the development of anti-infectives against the human pathogens, malaria or tuberculosis. We mapped the sequence conservation of 1-deoxy-xylulose-5-phosphate synthase on the protein structure and analyzed it in comparison with previously identified druggable pockets. We provide a recent overview of known inhibitors of the enzyme. Taken together, this sets the stage for future structure-based drug design.

Structural insights into the alternative oxidases: are all oxidases made equal?

The alternative oxidases (AOXs) are ubiquinol-oxidoreductases that are members of the diiron carboxylate superfamily. They are not only ubiquitously distributed within the plant kingdom but also found in increasing numbers within the fungal, protist, animal and prokaryotic kingdoms. Although functions of AOXs are highly diverse in general, they tend to play key roles in thermogenesis, stress tolerance (through the management of radical oxygen species) and the maintenance of mitochondrial and cellular energy homeostasis. The best structurally characterised AOX is from Trypanosoma brucei In this review, we compare the structure of AOXs, created using homology modelling, from many important species in an attempt to explain differences in activity and sensitivity to AOX inhibitors. We discuss the implications of these findings not only for future structure-based drug design but also for the design of novel AOXs for gene therapy.

Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

The mitochondrial (mt) FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF), but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF), which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm). Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1) binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1), but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

Anti-influenza Virus Effects of Catechins: A Molecular and Clinical Review.

Influenza infection and associated epidemics represent a serious public health problem. Several preventive and curative measures exist against its spread including vaccination and therapeutic agents such as neuraminidase inhibitors (e.g., oseltamivir, zanamivir, as well as peramivir and laninamivir, which are licensed in several countries) and adamantanes (e.g., amantadine and rimantadine). However, neuraminidase inhibitor- and adamantane- resistant viruses have been detected, whereas vaccines exhibit strain-specific effects and are limited in supply. Thus, new approaches are needed to prevent and treat influenza infections. Catechins, a class of polyphenolic flavonoids present in tea leaves, have been reported as potential anti-influenza virus agents based on experimental and clinical studies. (-)-epigallocatechin gallate (EGCG), a major and highly bioactive catechin, is known to inhibit influenza A and B virus infections in Madin-Darby canine kidney cells. Additionally, EGCG and other catechin compounds such as epicatechin gallate and catechin-5-gallate also show neuraminidase inhibitory activities as demonstrated via molecular docking. These catechins can bind differently to neuraminidase and might overcome known drug resistancerelated virus mutations. Furthermore, the antiviral effects of chemically modified catechin derivatives have also been investigated, and future structure-based drug design studies of catechin derivatives might contribute to improvements in influenza prevention and treatment. This review briefly summarizes probable mechanisms underlying the inhibitory effects of tea catechins against influenza infection and their clinical benefits on influenza prevention and treatment. Additionally, the great potential of tea catechins and their chemical derivatives as effective antiviral agents is described.

Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion.

Polymerization of the cytosolic protein actin is critical to cell movement and host cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesized and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with that of latrunculin B against P. falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.

Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.

Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.