Legionella pneumophila is a facultative intracellular pathogen that causes Legionnaires’ disease or Pontiac fever in humans upon accidental inhalation of Legionella -contaminated aerosols. During infection, L. pneumophila secretes more than 300 effectors into the host for the biogenesis of a replication-permissive niche, known as the Legionella containing vacuole (LCV). Among these, a large number of effectors harbor protein domains that recognize specific phosphoinositide (PI) lipids and mediate the anchoring of these effectors to the surface of LCV or other host membrane-bound organelles. The Legionella effector SetA contains a unique C-terminal domain (SetA-CTD) that has been shown to specifically bind with phosphatidylinositol-3-phosphate (PI(3)P) and target SetA to endosomes and LCVs. Here, we report the NMR solution structure of SetA-CTD, which mainly comprises a four α-helix bundle. The structure reveals a basic pocket at one end of the α-helix bundle for PI(3)P binding and two hydrophobic loops for membrane insertion. Mutations of key residues involved in lipid binding result in the loss of SetA in membrane association and endosomal localization. Structural comparison with other PI(3)P-binding domains highlights a general theme applied by multiple families of phosphoinositide-binding domains across species.

An in vitro effect of (+)MK-801 (dizocilpine), an inhibitor of the glutamate/NMDA and nicotinic acetylcholine receptors, on the Aβ[1-42] and Aβ[1-40] peptides is described and compared to that of memantine. Memantine has been approved by the U.S. Food and Drug Administration for the treatment of mild-moderate Alzheimer's disease. Both compounds accelerated the formation of a β-sheet structure by Aβ[1-42], (+)MK-801 more rapidly than memantine, as observed in a thioflavin T fluorescence assay. The acceleration was followed by a decrease in the fluorescence signal that was not observed when the ligand was absent. Nuclear magnetic resonance spectra of the soluble peptides in the presence and absence of (+)MK-801 demonstrated that the monomeric form did not bind (+)MK-801 and that in the presence of (+)MK-801 the concentration of the monomeric form progressively decreased. Small angle X-ray scattering confirmed that the presence of (+)MK-801 resulted in a more rapid and characteristic transition to an insoluble form. These results suggest that (+)MK-801 and memantine accelerate the transition of Aβ[1-42] and Aβ[1-40] to ThT-negative insoluble forms.

The contamination of surface water and ground water by human activities, such as fossil fuel extraction and agriculture, can be difficult to assess due to incomplete knowledge of the chemicals and chemistry involved. This is particularly true for the potential contamination of drinking water by nearby extraction of oil and/or gas from wells completed by hydraulic fracturing. A case that has attracted considerable attention is unconventional natural gas extraction in Susquehanna County, Pennsylvania, particularly around Dimock, Pennsylvania. We analyzed surface water and groundwater samples collected throughout Susquehanna County with complementary biological assays and high-resolution mass spectrometry. We found that Ah receptor activity was associated with proximity to impaired gas wells. We also identified certain chemicals, including disclosed hydraulic fracturing fluid additives, in samples that were either in close proximity to impaired gas wells or that exhibited a biological effect. In addition to correlations with drilling activity, the biological assays and high-resolution mass spectrometry detected substances that arose from other anthropogenic sources. Our complementary approach provides a more comprehensive picture of water quality by considering both biological effects and a broad screening for chemical contaminants.

Glutamate is released from presynaptic nerve terminals in the central nervous system (CNS) and spreads excitation by binding to and activating postsynaptic iGluRs. Of the potential glutamate targets, tetrameric AMPA receptors mediate fast, transient CNS signaling. Each of the four AMPA subunits in the receptor channel complex is capable of binding glutamate at its ligand-binding domains and transmitting the energy of activation to the pore domain. Homotetrameric AMPA receptor channels open in a stepwise manner, consistent with independent activation of individual subunits, and they exhibit complex kinetic behavior that manifests as temporal shifts between four different conductance levels. Here, we investigate how two AMPA receptor-selective noncompetitive antagonists, GYKI-52466 and GYKI-53655, disrupt the intrinsic step-like gating patterns of maximally activated homotetrameric GluA3 receptors using single-channel recordings from cell-attached patches. Interactions of these 2,3-benzodiazepines with residues in the boundary between the extracellular linkers and transmembrane helical domains reorganize the gating behavior of channels. Low concentrations of modulators stabilize open and closed states to different degrees and coordinate the activation of subunits so that channels open directly from closed to higher conductance levels. Using kinetic and structural models, we provide insight into how the altered gating patterns might arise from molecular contacts within the extracellular linker-channel boundary. Our results suggest that this region may be a tunable locus for AMPA receptor channel gating.

Biotechnology and BioengineeringVolume 116, Issue 2 p. 235-238 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 116, Number 2, February 2019 First published: 07 January 2019 https://doi.org/10.1002/bit.26751AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Volume116, Issue2February 2019Pages 235-238 RelatedInformation

Postsynaptic AMPA/glutamate receptors, essential for neuronal excitability, are important targets for anticonvulsant therapy. This single channel study of the selective noncompetitive AMPA receptor antagonist, perampanel, was performed on homotetrameric GluA3 receptor-channels that open in a stepwise manner to four distinct conductance levels through independent subunit activation. Previous structural studies show that perampanel binds to four sites located within the extracellular/transmembrane boundary of closed AMPA receptor-channel subunits. We found that channels exposed to 1 or 2 μM perampanel opened mainly to the two lower conductance levels in a dose-dependent manner. Comparison of the single channel results in the structures of the full length AMPA receptor in the closed state bound to perampanel, and the open state provide insights into the mechanism of allosteric reduction of AMPA-receptor-mediated excitation in epilepsy.

Recombinant antigens exhibit targeted protectiveproperties and offer important opportunities in the development of therapeutic technologies. Biophysical and structural methods have become important tools for the rational design and engineering of improved antigen-based vaccines. Vaccines containing Leptospira immunoglobulin-like (Lig) protein-derived antigens are currently the most promising candidates for protective immunity against the globally prevalent bacterial pathogen, Leptospira interrogans; however, vaccine trials using these domains have produced inconsistent results. Here, we compare the thermostability of domains from the main immunogenic regions from major leptospiral antigens, LigA and LigB. By measuring temperature-dependent fluorescence decay of the hydrophobic core tryptophan, 17 individual Lig protein immunoglobulin-like (Ig-like) domains were shown to display a broad range of unfolding temperatures. For a majority of the domains, stability issues begin to occur at physiologically relevant temperatures. A set of chimeric Ig-like domains was used to establish the ability of transplanted domain regions to enhance thermostability. Further insights into the determinants for domain stabilization were explored with nuclear magnetic resonance dynamics and mutational analysis. The current study has yielded a set of thermostable Ig-like domain scaffolds for use in engineering antigen-based vaccines and demonstrates the importance of incorporating thermostability screening as a design parameter.

Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection. These obligatory structures exposed to the host immune system provide important targets for rational vaccine design. Here, we use a systematically designed series of multi-domain constructs in combination with small angle X-ray scattering (SAXS) to determine the structure of the main immunoreactive region from a major antigen from Leptospira interrogans, LigB. An anti-LigB monoclonal antibody library exhibits cell binding and bactericidal activity with extensive domain coverage complementing the elongated architecture observed in the SAXS structure. Combining antigenic motifs in a single-domain chimeric immunoglobulin-like fold generated a vaccine that greatly enhances leptospiral protection over vaccination with single parent domains. Our study demonstrates how understanding an antigen's structure and antibody accessible surfaces can guide the design and engineering of improved recombinant antigen-based vaccines.

Calcium-dependent inactivation (CDI) is an important mechanism that is common to a variety of ion channels, and is particularly important to the function of the N-methyl-D-aspartic acid (NMDA) receptor (1Ehlers M.D. Zhang S. Huganir R.L. et al.Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit.Cell. 1996; 84: 745-755Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 2Krupp J.J. Vissel B. Westbrook G.L. et al.Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.J. Neurosci. 1999; 19: 1165-1178Crossref PubMed Google Scholar). The NMDA receptor is a class of glutamate receptor that is a mediator of synaptic plasticity and has been a target for neurological drug development. In particular, spike timing-dependent plasticity is thought to be influenced by CDI (3Urakubo H. Honda M. Kuroda S. et al.Requirement of an allosteric kinetics of NMDA receptors for spike timing-dependent plasticity.J. Neurosci. 2008; 28: 3310-3323Crossref PubMed Scopus (52) Google Scholar). CDI is mediated by the interaction of calmodulin with a sequence on the intracellular C-terminal domain (C0) of the NMDA receptor 1 (NR1) subunit of the NMDA receptor (4Zhang S. Ehlers M.D. Huganir R.L. et al.Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors.Neuron. 1998; 21: 443-453Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar) and by the binding of calcium to calmodulin. Although the general outlines of the mechanism of CDI have been well known for over 20 years, the important details of the process have been lacking, in particular the time course of CDI after NMDA receptor activation. Reported in this issue of Biophysical Journal, Iacobucci and Popescu (5Iacobucci G.J. Popescu G.K. Resident calmodulin primes NMDA receptors for Ca2+-dependent inactivation.Biophys. J. 2017; (Published online July 14, 2017)https://doi.org/10.1016/j.bpj.2017.06.035Abstract Full Text Full Text PDF Scopus (19) Google Scholar) have designed a series of clever experiments that shed new light on this important process. The goals of their study were to define precisely the kinetics of CDI, to localize the calcium sensor (calmodulin) relative to the ion channel pore, and to determine if calmodulin binds to NR1 in the apo or holo form. The key to these measurements was a robust definition of CDI. This involved characterizing and excluding Ca2+-independent sources of inhibition. The other major Ca2+-dependent inhibition at the external DRPEER motif did not contribute to the results because the time course of inhibition at this site is rapid, present at time zero in the CDI measurements, and, thus, subtracted from the time course. In addition, a heterologous human embryonic kidney cell (HEK293) expression system for NR1-2a/NR2A receptors and calmodulin was used to facilitate independent transfection of various forms of calmodulin and NMDA receptors. By using this system, it became clear that endogenous calmodulin is generally insufficient to produce high levels of CDI. The greatest extent of CDI (overexpression of calmodulin) is ∼80%, with a residual activity of ∼20%. This suggests that CDI can be cell- and state-dependent and potentially affected by calmodulinopathies. Using these metrics, the time constant of the onset of CDI was 500 ms and recovery was 9.3 s. This is an important finding because, although the equilibrium level of CDI would not be reached during a single synaptic event, it would be significant during that time and multiple closely timed events would add to the degree of CDI. This provides a more dynamic, activity-dependent tuning of the postsynaptic signal. The next question was to determine the location of calmodulin relative to the mouth of the channel. This involved estimating an average unitary Ca2+ flux for a given external Ca2+ concentration and then using that to produce a relationship between CDI and steady-state Ca2+ flux (half the greatest level of CDI at 0.024 pA of Ca2+ current). Using this finding and employing Fick’s law, the authors were able to calculate the distance (∼9 nm) from the mouth of the channel to the site of action, presumably calmodulin. It is well known that calmodulin can interact with other proteins in either its apo or holo (calcium-bound) form. If bound to NR1 in the apo form, calmodulin would be primed to sense calcium influx through the channel. Although in vitro measurements clearly show that both apo and holo calmodulin can interact with the C0 region of NR1, with a higher affinity for the holo than the apo form (6Akyol Z. Bartos J.A. Hell J.W. et al.Apo-calmodulin binds with its C-terminal domain to the N-methyl-D-aspartate receptor NR1 C0 region.J. Biol. Chem. 2004; 279: 2166-2175Crossref PubMed Scopus (37) Google Scholar), it was not clear whether the receptor was primed for CDI by prebound apo-calmodulin or if Ca2+ was bound to calmodulin that then bound to C0, thus producing CDI. The authors derived predictions for the relationship between CDI and the po (probability of being in the open state) for each of these two models. Unfortunately, with native affinity of calmodulin for the NR1 C0 and the maximum po of wild-type NR1a/N2A receptors (0.62), either model could explain the data; however, the predicted holo calmodulin affinity for NR1 was lower than expected. By using a double mutation that lowered the calmodulin affinity and increased channel po, the data strongly supported the model favoring prebound apo-calmodulin. Although many pieces of this puzzle were known before this study, this whole-cell model of calmodulin action provides a comprehensive picture of CDI. As shown in Fig. 1, calmodulin is prebound to the C-terminal C0 region of NR1 ∼9 nm from the mouth of the channel. Given the considerable intracellular buffering of Ca2+, it is the influx of calcium through the channel after activation (binding of glutamate to NR2 and glycine to NR1) that increases the local concentration sufficiently to bind calmodulin and trigger CDI. This occurs with a time course of 500 ms. Glutamate unbinds (deactivation) and then Ca2+ dissociates from calmodulin with a time constant of 9.3 s. Although this study represents a notable increase in our understanding of CDI, this is not the end of the story. Previous work (7Merrill M.A. Malik Z. Hell J.W. et al.Displacement of alpha-actinin from the NMDA receptor NR1 C0 domain By Ca2+/calmodulin promotes CaMKII binding.Biochemistry. 2007; 46: 8485-8497Crossref PubMed Scopus (36) Google Scholar) has suggested that the activation of bound calmodulin by Ca2+ displaces α-actinin, which in turn allows the binding of Ca2+/calmodulin-dependent kinase II (CaMKII). The subsequent molecular events leading to CDI remain open questions. Various other questions remain as well. For example, is there any cross talk between NMDA receptors or is each receptor sensitive to only Ca2+ flux through its own channel? Are the two Ca2+ binding sites on calmodulin equivalent with respect to CDI? Why is the greatest extent of CDI 80%; are some receptors insensitive or is gating impaired by CDI but still present? What is the exact physiological role of CDI and is it neuroprotective in some neurological diseases? Future single molecule experiments using calmodulin mutations in combination with structure-dynamic investigations may shed light on some of these questions. Resident Calmodulin Primes NMDA Receptors for Ca2+-Dependent InactivationIacobucci et al.Biophysical JournalJuly 13, 2017In BriefN-methyl-d-aspartate (NMDA) receptors are glutamate- and glycine-gated channels that flux Na+ and Ca2+ into postsynaptic neurons during synaptic transmission. The resulting intracellular Ca2+ transient is essential to physiological and pathological processes related to synaptic development, plasticity, and apoptosis. It also engages calmodulin (CaM) to reduce subsequent NMDA receptor activity in a process known as Ca2+-dependent inactivation (CDI). Here, we used whole-cell electrophysiology to measure CDI and computational modeling to dissect the sequence of events that underlies it. Full-Text PDF Open Archive

We report here the synthesis of 7-phenoxy-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides and their evaluation as AMPA receptor positive allosteric modulators (AMPApams). The impact of substitution on the phenoxy ring and on the nitrogen atom at the 4-position was examined. At GluA2(Q) expressed in HEK293 cells (calcium flux experiment), the most potent compound was 11m (4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, EC50 = 2.0 nM). The Hill coefficient in the screening and the shape of the dimerization curve in small-angle X-ray scattering (SAXS) experiments using isolated GluA2 ligand-binding domain (GluA2-LBD) are consistent with binding of one molecule of 11m per dimer interface, contrary to most benzothiadiazine dioxides developed to date. This observation was confirmed by the X-ray structure of 11m bound to GluA2-LBD and by NMR. This is the first benzothiadiazine dioxide AMPApam to reach the nanomolar range.

Neurotransmitter receptor channels, such as ionotropic glutamate receptors, are proteins whose functional activity requires specific motions in response to the binding of small ligands. In order to understand how these proteins work, it is important not only to know the three-dimensional structure at atomic resolution but also to understand, at the atomic level, these characteristic motions. An important strength of the analysis of proteins with NMR spectroscopy is the detection of motions of individual atoms on a broad variety of timescales. The drawback is that relatively large concentrations of protein are required and, to a large extent, the size of the complex can be limiting. We describe here the production and isotopic labeling of the ligand-binding domain of an AMPA receptor (GluA2) to permit studies of protein dynamics by NMR. The strategies for resonance assignment and the experiments used to study dynamics are also described.

The kinetics of AMPA receptor activation, deactivation, and desensitization shape the synaptic current at a majority of synapses in the central nervous system; however, the sequence of molecular events that make up these receptors’ reaction mechanism is unknown. Information about reaction mechanism is best extracted from single-molecule observations, which for ion channels can be done in real time with the patch-clamp technique. AMPA receptors have complex single-channel behaviors, which have been difficult to organize into a comprehensive mechanism. Here we describe methodology to obtain lengthy recordings of currents from one AMPA receptor and show how kinetic analysis was applied to organize the signal into classes of activity or modes that follow similar kinetic patterns. This approach can provide valuable insights into the molecular transitions that produce the activation, deactivation, and desensitization of ionotropic glutamate receptors and of ligand-gated channels in general.

Positive allosteric modulators of AMPA receptors (GluAs) stabilize the activated GluA structure by binding to the glutamate-binding domain (LBD) dimer interface. By adding contacts across the dimer interface, allosteric modulators strengthen the LBD dimer and inhibit desensitization-linked dimer disruption. Structurally diverse molecules can occupy the interface to fill various pockets and act as LBD dimerizers. Functionally, allosteric modulators exert a primary allosteric effect by stabilizing the open channel; however, allosteric modulators with different dimer interface binding patterns lead to differing impacts on agonist site binding affinity. Here, we examine the secondary allosteric effects on the glutamate-binding site. Using nuclear magnetic resonance (NMR) spectroscopy, we observed the global backbone changes on the isolated GluA2 LBD as a result of allosteric modulator binding. Spectral changes at the dimer interface correlate well with allosteric modulator binding sites on bound X-ray crystal structures. The backbone residues near the agonist-binding site are also affected differently depending on the orientation of allosteric modulator binding. Further, allosteric modulator binding to the LBD induces local environmental changes at the agonist site as reported by NMR active nuclei on the LBD-bound agonist. By improving the molecular understanding of GluA allosteric modulators in the context of secondary agonist-site effects, our results provide further guidance for rational drug design.

Public health concerns related to the expansion of unconventional oil and gas drilling have sparked intense debate. In 2012, we published case reports of animals and humans affected by nearby drilling operations. Because of the potential for long-term effects of even low doses of environmental toxicants and the cumulative impact of exposures of multiple chemicals by multiple routes of exposure, a longitudinal study of these cases is necessary. Twenty-one cases from five states were followed longitudinally; the follow-up period averaged 25 months. In addition to humans, cases involved food animals, companion animals and wildlife. More than half of all exposures were related to drilling and hydraulic fracturing operations; these decreased slightly over time. More than a third of all exposures were associated with wastewater, processing and production operations; these exposures increased slightly over time. Health impacts decreased for families and animals moving from intensively drilled areas or remaining in areas where drilling activity decreased. In cases of families remaining in the same area and for which drilling activity either remained the same or increased, no change in health impacts was observed. Over the course of the study, the distribution of symptoms was unchanged for humans and companion animals, but in food animals, reproductive problems decreased and both respiratory and growth problems increased. This longitudinal case study illustrates the importance of obtaining detailed epidemiological data on the long-term health effects of multiple chemical exposures and multiple routes of exposure that are characteristic of the environmental impacts of unconventional drilling operations.

AMPA receptors (GluAs) are essential neuronal ligand-gated ion channels involved in learning and memory. The dimeric conformation of the GluA ligand-binding domain is involved in the coupling of agonist binding to channel gating. We have used NMR, crystallography, ITC, and single channel recording to study the mechanism of action of antagonists and partial agonists on the GluA2 receptor. Antagonists form stable but open cleft binding sites with little dynamics, with binding driven largely by enthalpy. On the other hand, considerable dynamics are observed in the binding site in the presence of partial agonists, whose binding, in most cases, has a large entropic component. Allosteric modulators bind to a large surface that is formed by the dimer interface of two ligand-binding domains in the resting and channel activated states. This binding prevents the dissociation of the dimer interface and inhibits desensitization of the receptor. The desensitized conformation is disrupted along the dimer interface; however, little is known about the dynamic equilibrium between the bound/dimerized form and the unbound/monomer forms. Using small angle x-ray scattering (SAXS), crystallography, and NMR spectroscopy, we developed an equilibrium model for modulator dependent dimerization. This model demonstrates that a second modulator-binding site produces both an increase in positive cooperativity and a higher apparent affinity. A combination of the crystal structures of the bound modulators and the binding model developed using SAXS data provide new clues for the development of more effective allosteric modulators that may have cognitive enhancing effects.

The extraction of hydrocarbons from shale formations using horizontal drilling with high volume hydraulic fracturing (unconventional shale gas and tight oil extraction), while derived from methods that have been used for decades, is a relatively new innovation that was introduced first in the United States and has more recently spread worldwide. Although this has led to the availability of new sources of fossil fuels for domestic consumption and export, important issues have been raised concerning the safety of the process relative to public health, animal health, and our food supply. Because of the multiple toxicants used and generated, and because of the complexity of the drilling, hydraulic fracturing, and completion processes including associated infrastructure such as pipelines, compressor stations and processing plants, impacts on the health of humans and animals are difficult to assess definitively. We discuss here findings concerning the safety of unconventional oil and gas extraction from the perspectives of public health, veterinary medicine, and food safety.

A number of surface proteins specific to pathogenic strains of Leptospira have been identified. The Lig protein family has shown promise as a marker in typing leptospiral isolates for pathogenesis and as an antigen in vaccines. We used NMR spectroscopy to solve the solution structure of the twelfth immunoglobulin-like (Ig-like) repeat domain from LigB (LigB-12). The fold is similar to that of other bacterial Ig-like domains and comprised mainly of β-strands that form a β-sandwich based on a Greek-key folding arrangement. Based on sequence analysis and conservation of structurally important residues, homology models for the other LigB Ig-like domains were generated. The set of LigB models illustrates the electrostatic differences between the domains as well as the possible interactions between neighboring domains. Understanding the structure of the extracellular portion of LigB and related proteins is important for developing diagnostic methods and new therapeutics directed toward leptospirosis.

Three residues within the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluA1 C terminus (Ser818, Ser831, Thr840) can be phosphorylated by Ca(2+)/phospholipid-dependent protein kinase (PKC). Here, we show that PKC phosphorylation of GluA1 Ser818 or Thr840 enhances the weighted mean channel conductance without altering the response time course or agonist potency. These data support the idea that these residues constitute a hyper-regulatory domain for the AMPA receptor. Introduction of phosphomimetic mutations increases conductance only at these three sites within the proximal C terminus, consistent with a structural model with a flexible linker connecting the distal C-terminal domain to the more proximal domain containing a helix bracketed by Ser831 and Thr840. NMR spectra support this model and raise the possibility that phosphorylation can alter the configuration of this domain. Our findings provide insight into the structure and function of the C-terminal domain of GluA1, which controls AMPA receptor function and trafficking during synaptic plasticity in the central nervous system.

Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with pKa values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery.