Structure-based Drug Design Program Research Articles

Recent Insights into the Structure and Function of Mycobacterial Membrane Proteins Facilitated by Cryo-EM.

Mycobacterium tuberculosis (Mtb) is one of the deadliest pathogens encountered by humanity. Over the decades, its characteristic membrane organization and composition have been understood. However, there is still limited structural information and mechanistic understanding of the constituent membrane proteins critical for drug discovery pipelines. Recent advances in single-particle cryo-electron microscopy and cryo-electron tomography have provided the much-needed impetus towards structure determination of several vital Mtb membrane proteins whose structures were inaccessible via X-ray crystallography and NMR. Important insights into membrane composition and organization have been gained via a combination of electron tomography and biochemical and biophysical assays. In addition, till the time of writing this review, 75 new structures of various Mtb proteins have been reported via single-particle cryo-EM. The information obtained from these structures has improved our understanding of the mechanisms of action of these proteins and the physiological pathways they are associated with. These structures have opened avenues for structure-based drug design and vaccine discovery programs that might help achieve global-TB control. This review describes the structural features of selected membrane proteins (type VII secretion systems, Rv1819c, Arabinosyltransferase, Fatty Acid Synthase, F-type ATP synthase, respiratory supercomplex, ClpP1P2 protease, ClpB disaggregase and SAM riboswitch), their involvement in physiological pathways, and possible use as a drug target.Graphic Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. The Cryo-EM and tomography have simplified the understanding of the mycobacterial membrane organization. Some proteins are located in the plasma membrane; some span the entire envelope, while some, like MspA, are located in the mycomembrane. Cryo-EM has made the study of such membrane proteins feasible.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00232-021-00179-w.

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.

A Chemoproteomic Strategy for Direct and Proteome-Wide Covalent Inhibitor Target-Site Identification.

Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.

Reviving B-Factors: Activating ALK Mutations Increase Protein Dynamics of the Unphosphorylated Kinase.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and nonphosphorylated wildtype and mutant ALK kinase domain was done. Our structure-based drug design programs directed at ALK allowed us to interrogate whether X-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics. Crystallographic B-factors were converted to normalized B-factors, which allowed analysis of wildtype ALK, ALK-C1156Y, and ALK-L1196M. This data suggests that mobility of the P-loop, αC-helix, and activation loop (A-loop) may be important in catalytic activity increases, with or without phosphorylation. Both molecular dynamics simulations and hydrogen-deuterium exchange experimental data corroborated the normalized B-factors data.

Tamoxifen Directly Interacts with the Dopamine Transporter.

The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 μM) and amphetamine-stimulated efflux (59% reduction at 10 μM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.

Improving virtual screening of G protein-coupled receptors via ligand-directed modeling.

G protein-coupled receptors (GPCRs) play crucial roles in cell physiology and pathophysiology. There is increasing interest in using structural information for virtual screening (VS) of libraries and for structure-based drug design to identify novel agonist or antagonist leads. However, the sparse availability of experimentally determined GPCR/ligand complex structures with diverse ligands impedes the application of structure-based drug design (SBDD) programs directed to identifying new molecules with a select pharmacology. In this study, we apply ligand-directed modeling (LDM) to available GPCR X-ray structures to improve VS performance and selectivity towards molecules of specific pharmacological profile. The described method refines a GPCR binding pocket conformation using a single known ligand for that GPCR. The LDM method is a computationally efficient, iterative workflow consisting of protein sampling and ligand docking. We developed an extensive benchmark comparing LDM-refined binding pockets to GPCR X-ray crystal structures across seven different GPCRs bound to a range of ligands of different chemotypes and pharmacological profiles. LDM-refined models showed improvement in VS performance over origin X-ray crystal structures in 21 out of 24 cases. In all cases, the LDM-refined models had superior performance in enriching for the chemotype of the refinement ligand. This likely contributes to the LDM success in all cases of inhibitor-bound to agonist-bound binding pocket refinement, a key task for GPCR SBDD programs. Indeed, agonist ligands are required for a plethora of GPCRs for therapeutic intervention, however GPCR X-ray structures are mostly restricted to their inactive inhibitor-bound state.

Enzymatic Characterization of Fructose 1,6-Bisphosphatase II from Francisella tularensis, an Essential Enzyme for Pathogenesis

The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis, and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF1631c). Our complementation work independently verifies that the glpX gene (FTF1631c) in F. tularensis is indeed an FBPase and supports the growth of the ΔglpX E. coli mutant on glycerol-containing media. We have performed heterologous expression and purification of the glpX encoded FBPaseII in F. tularensis. We have confirmed the function of glpX as an FBPase and optimized the conditions for enzymatic activity. Mn2+ was found to be an absolute requirement for activity, with no other metal substitutions rendering the enzyme active. The kinetic parameters for this enzyme were found as follows: Km 11 μM, Vmax 2.0 units/mg, kcat 1.2 s−1, kcat/Km 120 mM−1 s−1, and a specific activity of 2.0 units/mg. Size exclusion data suggested an abundance of a tetrameric species in solution. Our findings on the enzyme’s properties will facilitate the initial stages of a structure-based drug design program targeting this essential gene of F. tularensis.

Desmethyl macrolides: synthesis and evaluation of 4-desmethyl telithromycin.

Novel sources of antibiotics are needed to address the serious threat of bacterial resistance. Accordingly, we have launched a structure-based drug design program featuring a desmethylation strategy wherein methyl groups have been replaced with hydrogens. Herein we report the total synthesis, molecular modeling, and biological evaluation of 4-desmethyl telithromycin (6), a novel desmethyl analogue of the third-generation ketolide antibiotic telithromycin (2) and our final analogue in this series. While 4-desmethyl telithromycin (6) was found to be equipotent with telithromycin (2) against wild-type bacteria, it was 4-fold less potent against the A2058G mutant. These findings reveal that strategically replacing the C4-methyl group with hydrogen (i.e., desmethylation) did not address this mechanism of resistance. Throughout the desmethyl series, the sequential addition of methyls to the 14-membered macrolactone resulted in improved bioactivity. Molecular modeling methods indicate that changes in conformational flexibility dominate the increased biological activity; moreover, they reveal 6 adopts a different conformation once bound to the A2058G ribosome, thus impacting noncovalent interactions reflected in a lower MIC value. Finally, fluorescence polarization experiments of 6 with E. coli ribosomes confirmed 6 is indeed binding the ribosome.

Applications of structure-based design to antibacterial drug discovery

In recent years bacterial resistance has been observed against many of our current antibiotics, for instance most worryingly against the cephalosporins which are typically the last line of defence against many bacterial infections. Additionally the failure of high throughput screening in the discovery of new antibacterial drug leads has led to a decline in the number of antibacterial agents reaching the market. Alternative methods of drug discovery including structure based drug design are needed to meet the threats caused by the emergence of resistance. In this review we explore the latest advancements in the identification of new antibacterial agents through the use of a number of structure based drug design programs.

Desmethyl Macrolides: Synthesis and Evaluation of 4,8-Didesmethyl Telithromycin

There is an urgent need for novel sources of antibiotics to address the incessant and inevitable onset of bacterial resistance. To this end, we have initiated a structure-based drug design program that features a desmethylation strategy (i.e., replacing methyl groups with hydrogens). Herein we report the total synthesis, molecular modeling and biological evaluation of 4,8-didesmethyl telithromycin (5), a novel desmethyl analogue of the third-generation ketolide antibiotic telithromycin (2), which is an FDA-approved semisynthetic derivative of erythromycin (1). We found 4,8-didesmethyl telithromycin (5) to be eight times more active than previously prepared 4,8,10-tridesmethyl congener (3) and two times more active than 4,10-didesmethyl regioisomer (4) in MIC assays. While less potent than telithromycin (2) and paralleling the observations made in the previous study of 4,10-didesmethyl analogue (4), the inclusion of a single methyl group improves biological activity thus supporting its role in antibiotic activity.

Natural Products of Dietary Origin as Lead Compounds in Virtual Screening and Drug Design

Natural products have been found to be useful in the treatment of several diseases across the ages. In this article, we review the use of natural products, obtained from dietary sources, as lead compounds in developing novel therapeutic agents. These compounds have shown tremendous promise in the prevention and as well as treatment of a variety of chronic ailments. In addition, to being patentable and biocompatible, these compounds are a rich source of novel scaffolds to invigorate the pipelines of the pharmaceutical industry. In this communication, we also focus on studies which show how natural products have proved useful as lead compounds in virtual screening and structure-based drug design programs. Natural dietary constituents, such as resveratrol, curcumin and caffeine as well as other compounds, are discussed to illustrate this approach.

Adsorption of xenon on a protein arising from the translational motion of solvent molecules

A simple method was used to predict binding sites and to calculate the binding free energy for a xenon atom on a protein to determine the importance of the translational motion of water molecules in molecular recognition. We examined xenon bound on myoglobin and on a fragment of ammonium transporter. Despite the simplicity of our method, the predicted binding sites and the experimental results agree very well, and the estimated values of the free energy gain are also reasonable. We discuss the van der Waals picture of molecular recognition between a protein and a small hydrophobic molecule, such as an anesthetic molecule, which gives us a simple physical justification for the idea of a "lock and key" relationship used in conventional structure-based drug design programs.

Abstract A142: Molecular modeling approach to the rational design of promiscuous quinazoline-based EGFR inhibitors

Abstract Solid tumors at the advanced stages are often characterized by the overexpression of tyrosine kinase receptors that stimulate growth through the MAP kinase pathway and activate antiapoptotic signaling through the PI3K kinase pathway. One such receptor is the epidermal growth factor receptor (EGFR) that is overexpressed in many tumors including brain, lung, breast, ovarian and prostate carcinomas. EGFR can synergize with other tyrosine kinases such as Src to promote invasion and metastasis. However, despite the implication of EGFR in multiple processes, inhibitors of its tyrosine kinase activity such as ZD1839 or erlotinib showed moderate antiproliferative activity against certain tumor types in the clinic. It has been reported that EGFR expressing cells that harbor dysfunctional PTEN, are moderately sensitive to EGFR inhibitors. Given the complexity of cell response the latter class of inhibitors, we thought it of interest to investigate agents that are not only directed at EGFR but also to divergent targets such as Src or DNA, with the purpose of producing single compounds with greater potency than their single inhibitor counterpart. These molecules termed “combi-molecules” were designed to remain small enough for their quinazoline moiety to be able to bind to the ATP site of EGFR and to block a divergent target such as Src or DNA. Using molecular modeling, a structure-based drug design program was used to identify a linker that could be placed between the quinazoline moiety required for binding in the ATP site and the appendage directed at the divergent target. A solvent exposed ASP residue near the opening of the EGFR ATP binding pocket was used to optimize interaction with ionizable linkers. This interaction was found to be tolerant of bulky substituents and was exploited to append other pharmacophores to the combi-molecules. The results showed that in the category of mixed EGFR-DNA targeting molecules, the EGFR inhibitory potency of EGFR was in the low micromolar to nM range and the compounds also retained significant DNA damaging potential. In the category of EGFR-Src targeting molecules, we identified SB163 that contained a quinazoline moiety, the ionizable spacer and an analogue of PP2. SB163 showed significant antiproliferative and antimetastatic property in a Boyden Chamber assay and its activity was superior to that of a combination of known Src inhibitor PP2 + ZD1839, a clinical inhibitor of EGFR. Furthermore, sub-cellular distribution studies, using a fluorescent probe containing the optimized ionizable spacer showed that their biodistribution is unique with preferential localization in the perinuclear region. The results in toto suggests that in the design of promiscuous molecules, an ionizable basic arm linked to the aminoquinazoline moiety preserves EGFR inhibitory potency and allow freedom to append other moiety directed at the cross- target. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A142.

X‐ray structures of Sap1 and Sap5: Structural comparison of the secreted aspartic proteinases from Candida albicans

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.

A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution.

Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 A resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration.

High‐Throughput Protein Crystallography and Drug Discovery

AbstractFor Abstract see ChemInform Abstract in Full Text.

Studying protein–ligand interactions using protein crystallography

Protein X-ray crystallography is now a relatively high-throughput technique that plays a major role in structure-based drug design programs where the method of soaking small organic ligands into protein crystals is used to confirm or identify binding modes. In this review, we discuss some experimental approaches for ‘soaking-in’ ligands into protein crystals where the major problem is poor solubility of the ligand. We also provide examples showing how crystal soaking can be used as a technique for establishing ligand binding strength: K c (the apparent ligand–protein dissociation constant in the crystal), which for some proteins at least, is found to be very similar to the solution K d. Kinetic effects are also found to be important and the rate at which ligands soak into crystals is shown to vary by orders of magnitude from 10−1 to 107 s depending on the system.

High Resolution Crystal Structure of Human Rab9 GTPase: A NOVEL ANTIVIRAL DRUG TARGET

Rab GTPases and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome to trans-Golgi transport and has recently been found to be a key cellular component for human immunodeficiency virus-1, Ebola, Marburg, and measles virus replication, suggesting that it may be a novel target in the development of broad spectrum antiviral drugs. As part of our structure-based drug design program, we have determined the crystal structure of a C-terminally truncated human Rab9 (residues 1-177) to 1.25-A resolution. The overall structure shows a characteristic nucleotide binding fold consisting of a six-stranded beta-sheet surrounded by five alpha-helices with a tightly bound GDP molecule in the active site. Structure-based sequence alignment of Rab9 with other Rab proteins reveals that its active site consists of residues highly conserved in the Rab GTPase family, implying a common catalytic mechanism. However, Rab9 contains seven regions that are significantly different in conformation from other Rab proteins. Some of those regions coincide with putative effector-binding sites and switch I and switch II regions identified by structure/sequence alignments. The Rab9 structure at near atomic resolution provides an excellent model for structure-based antiviral drug design.

Refinement of the conformation of UDP-galactose bound to galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization.

The STD NMR technique has originally been described as a tool for screening large compound libraries to identify the lead compounds that are specific to target proteins of interest. The application of this technique in the qualitative epitope mapping of ligands weakly binding to proteins, virus capsid shells, and nucleic acids has also been described. Here we describe the application of the STD NMR intensity-restrained CORCEMA optimization (SICO) procedure for refining the bound conformation of UDP-galactose in galactosyltransferase complex using STD NMR intensities recorded at 500 MHz as the experimental constraints. A comparison of the SICO structure for the bound UDP-galactose in solution with that in the crystal structure for this complex shows some differences in ligand torsion angles and V253 side-chain orientation in the protein. This work describes the first application of an STD NMR intensity-restrained CORCEMA optimization procedure for refining the torsion angles of a bound ligand structure. This method is likely to be useful in structure-based drug design programs since most initial lead compounds generally exhibit weak affinity (millimolar to micromolar) to target proteins of pharmaceutical interest, and the bound conformation of these lead compounds in the protein binding pocket can be determined by the CORCEMA-ST refinement.

Mark Murcko discusses enabling technologies in drug discovery

Mark is responsible for the strategic evaluation, identification, and integration of new technologies into worldwide R&D. He joined Vertex as a Founding Scientist in 1990 and started the company's molecular modeling, bioinformatics, IS and chemoinformatics groups. He is a co-inventor of a half-dozen compounds in Vertex's clinical pipeline. Notable among these are the HIV-protease inhibitors Agenerase® and Lexiva®, Vertex's first two marketed drugs, as well as the anti-inflammatory compounds Pralnacasan® and VX-765, both oral blockers of the IL-1 beta converting enzyme. Mark also played a key role in the creation and launch of Vertex's gene family approach, dubbed chemogenomics. Mark was named Chief Technology Officer in 2001. Prior to Vertex, Mark was at Merck in West Point, PA (1987–1990), where he helped discover multiple clinical candidates against cardiovascular and ocular diseases, including inhibitors of the enzyme carbonic anhydrase for the treatment of glaucoma. One of Merck's development candidates in this area, Trusopt®, became the first marketed drug in pharmaceutical history to result from a structure-based drug design program. Mark is a co-inventor on more than 30 issued patents, has co-authored more than 75 scientific articles, and has delivered more than 100 invited lectures. He currently sits on the scientific advisory boards of several outside companies and over the years has served as an editorial board member for Drug Discovery Today and the Journal of Medicinal Chemistry, among others.