Chemical Starting Points Research Articles

Screening a Natural Product-Based Library against Kinetoplastid Parasites.

Kinetoplastid parasites cause vector-borne parasitic diseases including leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease. These Neglected Tropical Diseases (NTDs) impact on some of the world’s lowest socioeconomic communities. Current treatments for these diseases cause severe toxicity and have limited efficacy, highlighting the need to identify new treatments. In this study, the Davis open access natural product-based library was screened against kinetoplastids (Leishmania donovani DD8, Trypanosoma brucei brucei and Trypanosoma cruzi) using phenotypic assays. The aim of this study was to identify hit compounds, with a focus on improved efficacy, selectivity and potential to target several kinetoplastid parasites. The IC50 values of the natural products were obtained for L. donovani DD8, T. b. brucei and T. cruzi in addition to cytotoxicity against the mammalian cell lines, HEK-293, 3T3 and THP-1 cell lines were determined to ascertain parasite selectivity. Thirty-one compounds were identified with IC50 values of ≤10 µM against the kinetoplastid parasites tested. Lissoclinotoxin E (1) was the only compound identified with activity across all three investigated parasites, exhibiting IC50 values <5 µM. In this study, natural products with the potential to be new chemical starting points for drug discovery efforts for kinetoplastid diseases were identified.

Screening the Medicines for Malaria Venture Pathogen Box across Multiple Pathogens Reclassifies Starting Points for Open-Source Drug Discovery.

ABSTRACTOpen-access drug discovery provides a substantial resource for diseases primarily affecting the poor and disadvantaged. The open-access Pathogen Box collection is comprised of compounds with demonstrated biological activity against specific pathogenic organisms. The supply of this resource by the Medicines for Malaria Venture has the potential to provide new chemical starting points for a number of tropical and neglected diseases, through repurposing of these compounds for use in drug discovery campaigns for these additional pathogens. We tested the Pathogen Box against kinetoplastid parasites and malaria life cycle stages in vitro. Consequently, chemical starting points for malaria, human African trypanosomiasis, Chagas disease, and leishmaniasis drug discovery efforts have been identified. Inclusive of this in vitro biological evaluation, outcomes from extensive literature reviews and database searches are provided. This information encompasses commercial availability, literature reference citations, other aliases and ChEMBL number with associated biological activity, where available. The release of this new data for the Pathogen Box collection into the public domain will aid the open-source model of drug discovery. Importantly, this will provide novel chemical starting points for drug discovery and target identification in tropical disease research.

Structure-based screening, ADMET profiling, and molecular dynamic studies on mGlu2 receptor for identification of newer antiepileptic agents

Structure-based screening approach targeting mGlu2 receptor was carried out to identify good chemical starting points for anti-epileptic therapy. Interactive modes of final 12 compounds identified on the basis of screening of Asinex library, binding energy analysis, ADME profiling with special emphasis for CNS ranges, and toxicity analysis were studied and showed good binding modes in the mGluR2-active site. Enrichment studies for validating screening protocol were carried out which gave ROC values 0.98 (AUC = 0.96) for SP, 0.97 (AUC = 0.95) for XP with BEDROC analysis. Our results indicate that all the 12 hits showed good CNS drug-like properties, have better binding free energy and ADME profile as compared to co-crystallized ligand with the best ligand hit retaining conserved hydrogen bond interactions with Ala-166, Thr-168, Ser-145, and Arg-61 residues in bilobatevenus fly-trap domain of mGluR2 receptor. Molecular dynamics simulations proved that the two potential hits, qualifying all screening parameters, are stable in the receptor active site pocket, confirming the potential of the identified hits as a specific target for mGluR2. Because the newly discovered mGluR2 agonists are structurally different with Tc values ranging from 0.57 to 0.92, all of them can be considered for further de novo design methods.

Structure-Based Identification of Inhibitory Fragments Targeting the p300/CBP-Associated Factor Bromodomain.

The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors.

A Rapid Python-Based Methodology for Target-Focused Combinatorial Library Design.

The chemical space is so vast that only a small portion of it has been examined. As a complementary approach to systematically probe the chemical space, virtual combinatorial library design has extended enormous impacts on generating novel and diverse structures for drug discovery. Despite the favorable contributions, high attrition rates in drug development that mainly resulted from lack of efficacy and side effects make it increasingly challenging to discover good chemical starting points. In most cases, focused libraries, which are restricted to particular regions of the chemical space, are deftly exploited to maximize hit rate and improve efficiency at the beginning of the drug discovery and drug development pipeline. This paper presented a valid methodology for fast target-focused combinatorial library design in both reaction-based and production-based ways with the library creating rates of approximately 70,000 molecules per second. Simple, quick and convenient operating procedures are the specific features of the method. SHAFTS, a hybrid 3D similarity calculation software, was embedded to help refine the size of the libraries and improve hit rates. Two target-focused (p38-focused and COX2-focused) libraries were constructed efficiently in this study. This rapid library enumeration method is portable and applicable to any other targets for good chemical starting points identification collaborated with either structure-based or ligand-based virtual screening.

Discovery of Subtype Selective Janus Kinase (JAK) Inhibitors by Structure-Based Virtual Screening.

Janus kinase inhibitors represent a promising opportunity for the pharmaceutical intervention of various inflammatory and oncological indications. Subtype selective inhibition of these enzymes, however, is still a very challenging goal. In this study, a novel, customized virtual screening protocol was developed with the intention of providing an efficient tool for the discovery of subtype selective JAK2 inhibitors. The screening protocol involves protein ensemble-based docking calculations combined with an Interaction Fingerprint (IFP) based scoring scheme for estimating ligand affinities and selectivities, respectively. The methodology was validated in retrospective studies and was applied prospectively to screen a large database of commercially available compounds. Six compounds were identified and confirmed in vitro, with an indazole-based hit exhibiting promising selectivity for JAK2 vs JAK1. Having demonstrated that the described methodology is capable of identifying subtype selective chemical starting points with a favorable hit rate (11%), we believe that the presented screening concept can be useful for other kinase targets with challenging selectivity profiles.

Pertamina's RFCC project at Cilicap currently in commissioning phase

High-throughput screening (HTS) is widely used in the pharmaceutical industry to identify novel chemical starting points for drug discovery projects. The current study focuses on the relationship between molecular hit rate in recent in-house HTS and four common molecular descriptors: lipophilicity (ClogP), size (heavy atom count, HEV), fraction of sp3-hybridized carbons (Fsp3), and fraction of molecular framework (fMF). The molecular hit rate is defined as the fraction of times the molecule has been assigned as active in the HTS campaigns where it has been screened. Beta-binomial statistical models were built to model the molecular hit rate as a function of these descriptors. The advantage of the beta-binomial statistical models is that the correlation between the descriptors is taken into account. Higher degree polynomial terms of the descriptors were also added into the beta-binomial statistic model to improve the model quality. The relative influence of different molecular descriptors on molecular hit rate has been estimated, taking into account that the descriptors are correlated to each other through applying beta-binomial statistical modeling. The results show that ClogP has the largest influence on the molecular hit rate, followed by Fsp3 and HEV. fMF has only a minor influence besides its correlation with the other molecular descriptors.

Virtual Screening for Transition State Analogue Inhibitors of IRAP Based on Quantum Mechanically Derived Reaction Coordinates.

Transition state and high energy intermediate mimetics have the potential to be very potent enzyme inhibitors. In this study, a model of peptide hydrolysis in the active site of insulin-regulated aminopeptidase (IRAP) was developed using density functional theory calculations and the cluster approach. The 3D structure models of the reaction coordinates were used for virtual screening to obtain new chemical starting points for IRAP inhibitors. This mechanism-based virtual screening process managed to identify several known peptidase inhibitors from a library of over 5 million compounds, and biological testing identified one compound not previously reported as an IRAP inhibitor. This novel methodology for virtual screening is a promising approach to identify new inhibitors mimicking key transition states or intermediates of an enzymatic reaction.

Abstract 99: Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™

Abstract Epigenetic processes control gene expression and play a major role in cell proliferation, survival and differentiation. Dysregulation of epigenetic mechanisms may lead to alterations in gene expression, resulting in cellular transformation and malignant outgrowth. Epigenetic enzymes are recognized as promising therapeutic targets for the treatment of cancer. Inventiva has several research programs in epigenetics, with specific focus on histone lysine methyltransferases (HKMTs). Emerging evidence suggests that G9a, a HKMT, able to mono and dimethylate H3K9 leading to the inactivation of tumor suppressor genes, plays a predominant role in lung and ovarian cancers. To identify novel G9a inhibitors, we used an AlphaLisa assay, with a histone H3-derived peptide and a specific antibody against H3K9me2. The assay, in 384-well format, displayed a robust Z-factor (0.84) with a signal to noise ratio around 800. We screened Inventiva's proprietary compound library using this assay. Three hundred thirty-six hits inhibited G9a by more than 60% (Hit rate: 0.14%). Despite the advantages offered by AlphaLisa technology, such as high S/N ratios and straightforward automation, this technology has been reported to generate a high number of false positives. In order to confirm the activity of our hits, we used a different technology, the methyltransferase Glo (MT-Glo) assay, which measures the reaction product SAH by luminescence read-out. Out of the 336 identified compounds, 75 were confirmed by this orthogonal screening (Hit confirmation rate: 22%). Additional compound characterization is ongoing to allow the selection of the best hit series for chemical optimization. In conclusion, the AlphaLisa assay, coupled with MT-Glo assay allowed the identification and confirmation of potent hits against G9a, as chemical starting points for our hit-to-lead optimization program. Citation Format: Claudia Fromond, Xavier Espanel, Laurent Chene, Philippe Masson, Benaïssa Boubia, Christian Montalbetti, Pierre Broqua. Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 99. doi:10.1158/1538-7445.AM2015-99

Abstract 105: A rational approach for the discovery of inhibitors of NSD2 for the treatment of cancer

Abstract Multiple myeloma (MM) is a plasma cell malignancy which accounts for approximately 10% of hematologic malignancies. Despite the introduction of new therapeutic agents, MM remains incurable and nearly all patients ultimately relapse. About 20% of MM are due to a chromosomal translocation t(4;14) leading to overexpression of the NSD2 histone methyltransferase. NSD2 catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Several studies have shown that in MM harboring the translocation t(4;14), oncogenic programming is dependent on the methyltransferase activity of NSD2. In addition, the NSD2 overactivity is also observed in prostate and lung cancers. Thus, NSD2 is a potential target for cancers, for which no selective drug is available to date. Using the AlphaLisa™ technology, we screened 240,000 compounds coming from our proprietary library. The assay is based on the detection of H3K36me2 on nucleosome by a specific antibody. False positives hits were removed by a technological counterscreen, and compounds with redox activity were excluded. This strategy allowed us to identify about 200 compounds. To focus on the most interesting ones, an orthogonal counterscreen based on 3H SAM incorporation is being completed. In parallel to the biochemical screening, we are developing secondary cellular assays based on the H3K36me2 methylation and proliferation to further confirm hit activity. To our knowledge, no NSD2 inhibitor have been identified to date despite several screening effort performed by other groups. Our library has already produced new chemical starting points for other KMTs, and we believe that our hits could be promising starting points to generate potent and selective NSD2 inhibitors. Citation Format: Claudia Fromond, Xavier Espanel, Anne Soudé, Laurent Chene, Philippe Masson, Benaïssa Boubia, Christian Montalbetti, Pierre Broqua. A rational approach for the discovery of inhibitors of NSD2 for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 105. doi:10.1158/1538-7445.AM2015-105

A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

DNA Encoded Library Selections and Insights Provided by Computational Simulations.

DNA encoded library (DEL) technology allows for rapid generation of extremely large numbers of small molecules and is often used to find novel chemical starting points for pharmaceutically relevant proteins. DEL selection output consists of a list of small-molecule structures and enrichment levels. It is widely presumed that molecules with greater enrichment will have larger equilibrium association constants, and follow-up efforts are triaged accordingly. Herein we describe a simple mathematical model used to simulate DEL selections. Simulations predict that enrichment levels will correlate poorly with equilibrium association constants when selections use high concentrations of protein or lower quality DELs (high variance in final product synthetic yields). A potentially superior technique is demonstrated to qualitatively assess equilibrium association constants directly from sequencing data. This technique requires conducting selections over a range of protein concentrations, so that the influence of synthetic yield can be accounted for.

Combinatorial Pharmacophore-Based 3D-QSAR Analysis and Virtual Screening of FGFR1 Inhibitors.

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway plays crucial roles in cell proliferation, angiogenesis, migration, and survival. Aberration in FGFRs correlates with several malignancies and disorders. FGFRs have proved to be attractive targets for therapeutic intervention in cancer, and it is of high interest to find FGFR inhibitors with novel scaffolds. In this study, a combinatorial three-dimensional quantitative structure-activity relationship (3D-QSAR) model was developed based on previously reported FGFR1 inhibitors with diverse structural skeletons. This model was evaluated for its prediction performance on a diverse test set containing 232 FGFR inhibitors, and it yielded a SD value of 0.75 pIC50 units from measured inhibition affinities and a Pearson’s correlation coefficient R2 of 0.53. This result suggests that the combinatorial 3D-QSAR model could be used to search for new FGFR1 hit structures and predict their potential activity. To further evaluate the performance of the model, a decoy set validation was used to measure the efficiency of the model by calculating EF (enrichment factor). Based on the combinatorial pharmacophore model, a virtual screening against SPECS database was performed. Nineteen novel active compounds were successfully identified, which provide new chemical starting points for further structural optimization of FGFR1 inhibitors.

Collaborative approaches for Target ID in neglected diseases: identifying new antimalarial targets

Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by P. falciparum. Most antimalarial drugs face decreased efficacy due to the spread of resistance. Most of the current compounds used for malaria treatment share related mechanisms of action so, there is an urgent need to identify new tractable targets to enable next generation of antimalarial drugs. The malaria drug discovery community has embraced phenotypic screening approaches as the strategy of choice for the generation of novel chemical starting points for Lead Optimization programs. Determining the mode of action for novel whole cell hits remains a challenge, but new technologies, including genetics, chemoinformatics and proteomics are proving successful. A chemical proteomics platform aimed at identifying antimalarial targets has been established. This platform is supported by The Bill & Melinda Gates Foundation and encompasses chemical probe synthesis, biological material preparation, pull down experiments and LC-MS protein-lead family characterization for chemical families of interest. Candidate scaffolds to be investigated through this platform are selected based on their biological profile by a join Steering Committee constituted by both GSK and BMGF members.

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

On the Relationship between Molecular Hit Rates in High-Throughput Screening and Molecular Descriptors

High-throughput screening (HTS) is widely used in the pharmaceutical industry to identify novel chemical starting points for drug discovery projects. The current study focuses on the relationship between molecular hit rate in recent in-house HTS and four common molecular descriptors: lipophilicity (ClogP), size (heavy atom count, HEV), fraction of sp3-hybridized carbons (Fsp3), and fraction of molecular framework (fMF). The molecular hit rate is defined as the fraction of times the molecule has been assigned as active in the HTS campaigns where it has been screened. Beta-binomial statistical models were built to model the molecular hit rate as a function of these descriptors. The advantage of the beta-binomial statistical models is that the correlation between the descriptors is taken into account. Higher degree polynomial terms of the descriptors were also added into the beta-binomial statistic model to improve the model quality. The relative influence of different molecular descriptors on molecular hit rate has been estimated, taking into account that the descriptors are correlated to each other through applying beta-binomial statistical modeling. The results show that ClogP has the largest influence on the molecular hit rate, followed by Fsp3 and HEV. fMF has only a minor influence besides its correlation with the other molecular descriptors.

Erratum: Antimalarial drug discovery — approaches and progress towards new medicines

Nature Reviews Microbiology 11, 849–862 (2013) In the above article, reference 69 was cited incorrectly at the end of the sentence “these compounds have been resynthesized and this 'malaria box' of potential chemical starting points can be obtained from the Medicines for Malaria Venture (MMV)” and this superfluous citation has now been deleted online.

Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum

BackgroundRecent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.MethodsThe approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4–7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.ResultsThe speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.ConclusionThe results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

Optimisation and Validation of a High Throughput Screening Compatible Assay to Identify Inhibitors of the Plasma Membrane Calcium ATPase Pump - a Novel Therapeutic Target for Contraception and Malaria

ATPases, which constitute a major category of ion transporters in the human body, have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. We have recently found that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) results in infertility in male mice due to a selective defect in sperm motility. In addition, recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determines the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimise and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF® Transcreener® ADP (TR-FRET) assay to screen a drug library. PMCA4 membrane microsomes were prepared from HEK293 cells overexpressing PMCA4. Western blot quantification revealed nearly nine-fold increased expression of PMCA4 compared to LacZ (control virus)-infected cells. Maximal PMCA4 microsomal activity was achieved in the TR-FRET assay with 15ng/μl microsomal concentration, 30-minute pre-incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1μM free-calcium. Finally a dose-response curve for carboxyeosin (a non-specific PMCA inhibitor) under optimised conditions showed significant PMCA4 inhibition. Upon confirmation that the assay was suitable for high-throughput screening, we have screened the ChemBioNet small molecule library (~21,000 compounds) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1,494 primary hits. We have optimised the HTRF® Transcreener® ADP assay for high-throughput screening to identify PMCA4 inhibitors. The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non-hormonal contraception or anti-malaria therapy.

Front‐Loading Natural‐Product‐Screening Libraries for log P: Background, Development, and Implementation

In the period from January 1981 to December 2010, 1068 small-molecule new chemical entities (NCEs) were introduced, of which ca. 34% are either a natural product or a close analogue. While this metric reflects the impact natural products have played in delivering new chemical starting points (leads) for the pharmaceutical industry, it does not capture the decline this approach has suffered over the last 20 years as the high-throughput screening (HTS) of pure compound libraries has become more popular. An impediment to natural-product drug discovery in the HTS paradigm is the lack of a clear strategy that enables front-loading of an extract or fraction's chemical constituents so that they are compliant with lead- and drug-like chemical space. To address this imbalance, an approach based on lipophilicity, as measured by clog P has been developed that, together with advances being made in isolation and structural elucidation, can afford natural product leads in timelines compatible with pure compound screening.