Medicinal Chemistry Approaches Research Articles

Relationships between hydrogen bonds and halogen bonds in biological systems.

The recent recognition that halogen bonding (XB) plays important roles in the recognition and assembly of biological molecules has led to new approaches in medicinal chemistry and biomolecular engineering. When designing XBs into strategies for rational drug design or into a biomolecule to affect its structure and function, we must consider the relationship between this interaction and the more ubiquitous hydrogen bond (HB). In this review, we explore these relationships by asking whether and how XBs can replace, compete against or behave independently of HBs in various biological systems. The complex relationships between the two interactions inform us of the challenges we face in fully utilizing XBs to control the affinity and recognition of inhibitors against their therapeutic targets, and to control the structure and function of proteins, nucleic acids and other biomolecular scaffolds.

INCB24360 (Epacadostat), a Highly Potent and Selective Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitor for Immuno-oncology.

A data-centric medicinal chemistry approach led to the invention of a potent and selective IDO1 inhibitor 4f, INCB24360 (epacadostat). The molecular structure of INCB24360 contains several previously unknown or underutilized functional groups in drug substances, including a hydroxyamidine, furazan, bromide, and sulfamide. These moieties taken together in a single structure afford a compound that falls outside of “drug-like” space. Nevertheless, the in vitro ADME data is consistent with the good cell permeability and oral bioavailability observed in all species (rat, dog, monkey) tested. The extensive intramolecular hydrogen bonding observed in the small molecule crystal structure of 4f is believed to significantly contribute to the observed permeability and PK. Epacadostat in combination with anti-PD1 mAb pembrolizumab is currently being studied in a phase 3 clinical trial in patients with unresectable or metastatic melanoma.

Plasma marker based hepatoprotective evaluation of some novel synthesized benzofluorenone analogues: A medicinal chemistry approach

In an extensive search program towards development of new hepatoprotective agents, a series of novel benzofluorenones have been synthesized. These newly synthesized benzofluorenone derivatives exhibited protection against carbon tetrachloride (CCl4)-induced hepatotoxicity. This activity was estimated by levels of biochemical parameters such as aspartate aminotransferase (AST), serum alanine transferase (ALT), serum alkaline phosphatase (SALP) and total proteins (TP) carried out by using regular assay kits.

Ligands of Therapeutic Utility for the Liver X Receptors.

Liver X receptors (LXRs) have been increasingly recognized as a potential therapeutic target to treat pathological conditions ranging from vascular and metabolic diseases, neurological degeneration, to cancers that are driven by lipid metabolism. Amidst intensifying efforts to discover ligands that act through LXRs to achieve the sought-after pharmacological outcomes, several lead compounds are already being tested in clinical trials for a variety of disease interventions. While more potent and selective LXR ligands continue to emerge from screening of small molecule libraries, rational design, and empirical medicinal chemistry approaches, challenges remain in minimizing undesirable effects of LXR activation on lipid metabolism. This review provides a summary of known endogenous, naturally occurring, and synthetic ligands. The review also offers considerations from a molecular modeling perspective with which to design more specific LXRβ ligands based on the interaction energies of ligands and the important amino acid residues in the LXRβ ligand binding domain.

Small molecules targeting glycogen synthase kinase 3 as potential drug candidates for the treatment of retinitis pigmentosa

Retinitis pigmentosa (RP) is an inherited retinal dystrophy that courses with progressive degeneration of retinal tissue and loss of vision. Currently, RP is an unpreventable, incurable condition. We propose glycogen synthase kinase 3 (GSK-3) inhibitors as potential leads for retinal cell neuroprotection, since the retina is also a part of the central nervous system and GSK-3 inhibitors are potent neuroprotectant agents. Using a chemical genetic approach, diverse small molecules with different potency and binding mode to GSK-3 have been used to validate and confirm GSK-3 as a pharmacological target for RP. Moreover, this medicinal chemistry approach has provided new leads for the future disease-modifying treatment of RP.

Discovery and optimization of selective inhibitors of protein arginine methyltransferase 5 by docking-based virtual screening.

Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT enzyme critical for diverse cellular processes and different types of cancers. Many efforts have been made to discover novel scaffold PRMT5 inhibitors. Herein, we report the discovery of DC_P33 as a hit compound of PRMT5 inhibitor, identified by molecular docking based virtual screening and 3H-labeled radioactive methylation assays. Structure-activity relationship (SAR) analysis was performed on the analogs of DC_P33 and then structural modifications were done to improve its activity. Among the derivatives, the compound DC_C01 displayed an IC50 value of 2.8 μM, and good selectivity toward PRMT1, EZH2 and DNMT3A. Moreover, DC_C01 exhibited anti-proliferation activities against Z-138, Maver-1, and Jeko-1 cancer cells with EC50 values of 12 μM, 12 μM, and 10.5 μM, respectively. Taken together, these results contribute to the development of specific inhibitors against PRMT5 and cancer therapy.

A Comprehensive Review of N-Heterocycles as Cytotoxic Agents.

Scientific community is striving to understand the role of heterocycles and fused heterocycles in drug discovery programme due to its impact on multi-drug resistance (MDR) of anticancer drugs. Architecting of various scaffolds for cancer treatment has become gradually increased in many years. Till now there is no treatment which is so proficient that it can cure the cancer from the roots. Hence, it is very necessary to design novel anticancer agents with minimum side effects. Synthesis of hybrids from natural leads is one of the rationale approaches in medicinal chemistry. It remains a big challenge to invent new efficient drugs to beat cancer. The design and synthesis of fused molecules as anticancer agents is one of the great innovations of modern era. In drug discovery archetype, a variety of heterocycles have been considered for the development of novel lead compounds. This article presents some recent advancements in the field of anticancer heterocyclic agents all around the world and also attracted the structure activity relationship along with the structure of the most promising molecules along with IC50 values against various human cancer cell lines.

H3B-8800, an Orally Bioavailable Modulator of the SF3b Complex, Shows Efficacy in Spliceosome-Mutant Myeloid Malignancies

Mutations in RNA splicing factors confer an alteration of function and are common in patients with myelodysplastic syndrome (MDS, ~45%), chronic myelomonocytic leukemia (CMML, ~60%), and acute myeloid leukemia (AML) derived from these conditions. Recent data suggest that spliceosome-mutant cells are preferentially sensitive to genetic or pharmacologic splicing modulation compared with wildtype (WT) counterparts. Here, we describe the discovery of H3B-8800, a potent and orally bioavailable modulator of the SF3b complex, and demonstrate efficacy in models of spliceosome mutant myeloid malignancies including a novel xenograft system for CMML.H3B-8800 was identified through a medicinal chemistry approach aimed at identifying compounds with preferential lethality in spliceosome mutant cells. Using a scintillation proximity assay, we demonstrated that H3B-8800 potently binds to SF3b complexes containing either WT or mutant SF3B1 protein. Consistent with this, H3B-8800 showed dose-dependent modulation of splicing in in vitro biochemical splicing assays and cellular pharmacodynamic assays. Selectivity of H3B-8800 for the SF3b complex was confirmed through observing resistance in cells expressing SF3B1R1074H, an SF3B1 mutation previously shown to confer resistance to natural product splicing modulators.In the above biochemical and cellular assays, H3B-8800 affected splicing similarly regardless of spliceosome genotype. However, preferential inhibition of in vitro cell growth was observed in isogenic AML cells with endogenous knock-in of SF3B1K700E or SRSF2P95H mutations compared to WT counterparts. In animals xenografted with SF3B1K700E knock-in K562 cells, oral H3B-8800 treatment demonstrated dose-dependent splicing modulation and inhibited tumor growth, while no therapeutic impact was seen in WT controls. Similarly, anti-leukemic efficacy and improved survival were observed with H3B-8800 treatment in mice transplanted with Srsf2P95H/MLL-AF9 mouse AML cells, a result not seen in Srsf2 WT/MLL-AF9 counterpart leukemias.To understand the preferential effects on spliceosome mutant cells, RNA-seq analysis of isogenic K562 cells treated with H3B-8800 was performed. H3B-8800 induced intron retention and exon skipping, however these effects were not global and introns preferentially retained by H3B-8800 were shorter and more GC-rich compared to those unaffected by drug (Figure A). Interestingly, a substantial number of genes experiencing intron retention with H3B-8800 themselves encoded spliceosome components (Figure B). This suggests that the preferential effect of H3B-8800 on spliceosome mutant cells is due to the exquisite dependency of these cells on normal expression of spliceosome proteins.Next we aimed to understand the therapeutic potential of H3B-8800 in the context of CMML due to the high frequency of SRSF2 mutations and the need for improved outcome in this disorder. To this end, we developed a xenotransplantation model through direct intrafemoral injection of CD34+ cells from CMML patients into "NSGS" mice: a variant of NSG mice that express human IL3, SCF and GM-CSF. We specifically focused on CMML with <10% marrow blasts. Surprisingly, injection of >200,000 CD34+ cells achieved robust engraftment for all patients (n=7) with rapid lethality (median of 39 days). In vivo H3B-8800 administration substantially reduced leukemic burden in spliceosome-mutant but not spliceosome-WT CMML PDX (Figure C). Furthermore, 2.2-fold reductions in immunophenotypically-defined leukemia initiating cells were seen with H3B-8800 versus vehicle treatment in spliceosome-mutant CMML compared with no change in those mice engrafted with spliceosome-WT CMML.These data identify a novel therapeutic approach with selective lethality in myeloid cells bearing a spliceosome mutation. Despite the essential nature of splicing, CMML/AML cells without a spliceosome mutation were less sensitive to H3B-8800 compared with potent eradication of mutant counterparts. These data demonstrate the therapeutic potential of splicing modulation in spliceosome mutant cancers and H3B-8800 is currently undergoing clinical evaluation in patients with MDS, AML and CMML. [Display omitted] DisclosuresBuonamici:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Caleb:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Karr:H3 Biomedicine: Employment. Liu:H3 Biomedicine: Employment. Meeske:H3 Biomedicine: Employment. Mizui:Eisai: Employment. Pazolli:H3 Biomedicine: Employment. Prajapati:H3 Biomedicine: Employment. Wang:Eisai: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.

Trans‐scleral delivery of novel anti‐angiogenic small molecule inhibitors of SRPK1

PurposeDevelopment of non‐invasive, novel therapies for wet age‐related macular degeneration (wAMD) and diabetic macular oedema (DME) is hindered by insufficient delivery to the retina. SRPK1 is a novel target that regulates VEGF‐A splicing. We used a rational medicinal chemistry approach to specifically design novel inhibitors with properties required for trans‐scleral delivery.MethodsFreshly enucleated porcine eyes were dissected, and eye tissue was clamped into a scaffold with inhibitors in the upper chamber facing the sclera. Tissue was dissected at 24 h and compound measured by mass spectrometry. Compounds were screened based on structure, potency, selectivity, molecular weight, cLogP and SAR to inform further development. Efficacy and PK were evaluated in vivo in C57/Bl6 mice. New Zealand White rabbits received 3× daily eye drop for 6 days then eyes were dissected, compounds analysed by mass spectrometry.ResultsPotent SRPK1 inhibitors had improved permeability ex vivo compared to pazopanib (SPHINX‐A 1.47 × 10−6 cm/s, Pazopanib 0.07 × 10−6 cm/s p &lt; 0.0005). Modifications of the R1 and R2 domains, based on crystal structure analysis, led to enhanced permeability (SPHINX‐B 3.46 cm/s*10−6). Permeability did not correlate with molecular weight or cLogP but could be affected by additional parameters such as melanin binding. SPHINX‐A was detected at 0.008% of the total applied dose (84 μg) and above its target IC50 value in rabbit eyes. SRPK1 inhibitor eye drops inhibited laser‐CNV (EC50s &lt; 0.5 μM, n = 6–8, p &lt; 0.05, One‐way ANOVA).ConclusionsWith increased specificity, potency and trans‐scleral permeability, novel SRPK1 inhibitors have potential to reach therapeutic levels in posterior eye segments following eye drop administration and improve treatment for patients with wAMD and DME.

Targeting Cannabinoid CB2 Receptors in the Central Nervous System. Medicinal Chemistry Approaches with Focus on Neurodegenerative Disorders.

Endocannabinoids activate two types of specific G-protein-coupled receptors (GPCRs), namely cannabinoid CB1 and CB2. Contrary to the psychotropic actions of agonists of CB1 receptors, and serious side effects of the selective antagonists of this receptor, drugs acting on CB2 receptors appear as promising drugs to combat CNS diseases (Parkinson's disease, Huntington's chorea, cerebellar ataxia, amyotrohic lateral sclerosis). Differential localization of CB2 receptors in neural cell types and upregulation in neuroinflammation are keys to understand the therapeutic potential in inter alia diseases that imply progressive neurodegeneration. Medicinal chemistry approaches are now engaged to develop imaging tools to map receptors in the living human brain, to develop more efficacious agonists, and to investigate the possibility to develop allosteric modulators.

Design, Synthesis, and Cytotoxic Evaluation of Novel Tubulysin Analogues as ADC Payloads.

The tubulysin class of natural products has attracted much attention from the medicinal chemistry community due to its potent cytotoxicity against a wide range of human cancer cell lines, including significant activity in multidrug-resistant carcinoma models. As a result of their potency, the tubulysins have become an important tool for use in targeted therapy, being widely pursued as payloads in the development of novel small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs). A structure-based and parallel medicinal chemistry approach was applied to the synthesis of novel tubulysin analogues. These efforts led to the discovery of a number of novel and potent cytotoxic tubulysin analogues, providing a framework for our simultaneous report, which highlights the discovery of tubulysin-based ADCs, including use of site-specific conjugation to address in vivo stability of the C-11 acetate functionality.

From Erythromycin to Azithromycin and New Potential Ribosome-Binding Antimicrobials.

Macrolides, as a class of natural or semisynthetic products, express their antibacterial activity primarily by reversible binding to the bacterial 50S ribosomal subunits and by blocking nascent proteins’ progression through their exit tunnel in bacterial protein biosynthesis. Generally considered to be bacteriostatic, they may also be bactericidal at higher doses. The discovery of azithromycin from the class of macrolides, as one of the most important new drugs of the 20th century, is presented as an example of a rational medicinal chemistry approach to drug design, applying classical structure-activity relationship that will illustrate an impressive drug discovery success story. However, the microorganisms have developed several mechanisms to acquire resistance to antibiotics, including macrolide antibiotics. The primary mechanism for acquiring bacterial resistance to macrolides is a mutation of one or more nucleotides from the binding site. Although azithromycin is reported to show different, two-step process of the inhibition of ribosome function of some species, more detailed elaboration of that specific mode of action is needed. New macrocyclic derivatives, which could be more potent and less prone to escape bacterial resistance mechanisms, are also continuously evaluated. A novel class of antibiotic compounds—macrolones, which are derived from macrolides and comprise macrocyclic moiety, linker, and either free or esterified quinolone 3-carboxylic group, show excellent antibacterial potency towards key erythromycin-resistant Gram-positive and Gram-negative bacterial strains, with possibly decreased potential of bacterial resistance to macrolides.

Abstract 1231: Novel chromene analogs as small-molecule microtubule destabilizers for the treatment of chemo-resistant ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological malignancy with a 5-year survival rate of less than 40%, primarily because of treatment failure due to emergence of chemo-resistance to standard agents. This indicates a dire need for new treatments to improve survival rates. The tubulin dynamics is a promising target for new chemotherapeutic agents. In continuation of our efforts, using medicinal chemistry approach, we recently identified small molecular inhibitors of tubulin polymerization called chromenes, as new ovarian anti-cancer agents. A set of chromene compounds were screened for anti-cancer effect on cisplatin sensitive (A2780, SKOV-3, TOV112D) and cisplatin resistant (OVCAR-3, A2780-cisR, TOV112D-cisR) ovarian cancer cells. In vitro cell viability was assessed by fluorescence based Alamar Blue assay. Our preliminary studies identified SP-6-27 (IC50 range: 0.102± 0.006 - 2.24±0.056 μM) and SP-6-37 (IC50 range: 0.258± 0.159 - 3.61±0.049 μM) as most potent chromene analogs. Both compounds exhibited a potent anticancer activity towards both cisplatin sensitive (SP-6-27 IC50: 0.14± 0.03 μM; SP-6-37 IC50: 1.35± 0.88 μM) and cisplatin resistant (SP-6-27 IC50: 0.81± 1.2 μM; SP-6-37 IC50: 2.09± 1.7 μM) cell lines. The compounds exhibited least cytotoxicity towards normal ovarian epithelial cells (SP-6-27- IC50: 83.35 ± 9.47 μM; SP-6-37- IC50: 75.77 ± 5.37 μM). Additionally, the analysis of apoptotic changes as determined by Annexin-V assay revealed an enhanced apoptosis in both cisplatin sensitive and resistant cells upon treatment with compounds SP-6-27 or SP-6-37. Together, the data demonstrates that ovarian cancer cell lines respond sensitively to SP-6-27 and SP-6-37, demonstrating that the chromene scaffold is effective in suppressing ovarian cancer cell growth. The findings indicate that the novel chromene analogs are a new class of chemotherapeutic agents that will offer advantages for the treatment of ovarian cancer. Citation Format: Arpita Kulshrestha, Safaa A. Ibrahim, Gajendra K. Katara, Renukadevi Patil, Shivaputra Patil, Kenneth D. Beaman. Novel chromene analogs as small-molecule microtubule destabilizers for the treatment of chemo-resistant ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1231.

Abstract 3513: Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides

Abstract We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Treatment of cancer cells with WE induced a reduction in the levels of the antiapoptotic proteins cFLIPL and cFLIPS, resulting in an increased activation of caspase-8 on subsequent TRAIL binding to its death receptors DR4 or DR5. The reduction in cFLIPL and cFLIPS was due to their destabilization and increased degradation by the proteasome. Interestingly, WE (a 17-beta-hydoxywithanolide, 17-BHW) was a far superior TRAIL sensitizer than more widely studied withaferin A (WFA) and its analogues, which lack the 17-beta-hydroxy group and bear an opposite side chain orientation, exhibit more promiscuous reactivity and are much more directly toxic to cells. Therefore, over 30 natural and semi-synthetic 17-BHWs were evaluated for their ability to promote death ligand-mediated cancer cell death. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Our studies identified several 17-BHWs that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells ACHN to TRAIL-mediated apoptosis. These more active 17-BHWs were also more efficient at reducing cellular levels of cFLIPL and cFLIPS and enhancing caspase-8 activation. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition acetoxylation at C-18, an alpha orientation of the lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as TRAIL sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to TRAIL-mediated apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation. Funded by FNLCR Contract HHSN261200800001E Citation Format: Alan D. Brooks, Ya-ming Xu, E. M. Kithsiri Wijeratne, Poonam Tewary, Curtis J. Henrich, Cheryl L. Thomas, A. A. Leslie Gunatilaka, Thomas J. Sayers. Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3513.

Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors – Part 3

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein 1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We observed that the MDR1 efflux ratio was correlated with the pKa value of the acidic moiety. The resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic acid hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral administration, increased urethral resistance in a dose-dependent manner.

Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors—Part 2

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood–brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H3)methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.

Kinetic Aspects of the Interaction between Ligand and G Protein-Coupled Receptor: The Case of the Adenosine Receptors.

Ligand-receptor binding kinetics is an emerging topic in the drug research community. Over the past years, medicinal chemistry approaches from a kinetic perspective have been increasingly applied to G protein-coupled receptors including the adenosine receptors (AR), which are involved in a plethora of physiological and pathological conditions. The study of ligand-AR binding kinetics offers room for detailed structure-kinetics relationships next to more traditional structure-activity relationships. Their combination may facilitate the triage of candidate compounds in hit-to-lead campaigns. Furthermore, kinetic studies also help in understanding AR allosterism. Allosteric modulation may yield a change in the activity and conformation of a receptor resulting from the binding of a compound at a site distinct from where the endogenous agonist adenosine binds. Hence, in this Review, we summarize available data and evidence for the binding kinetics of orthosteric and allosteric AR ligands. We hope this Review will raise awareness to consider the kinetic aspects of drug-target interactions on both ARs and other drug targets.

ChemInform Abstract: Development of Advanced Macrosphelides: Potent Anticancer Agents

AbstractReview: 44 refs.

Natural product discovery: past, present, and future.

Microorganisms have provided abundant sources of natural products which have been developed as commercial products for human medicine, animal health, and plant crop protection. In the early years of natural product discovery from microorganisms (The Golden Age), new antibiotics were found with relative ease from low-throughput fermentation and whole cell screening methods. Later, molecular genetic and medicinal chemistry approaches were applied to modify and improve the activities of important chemical scaffolds, and more sophisticated screening methods were directed at target disease states. In the 1990s, the pharmaceutical industry moved to high-throughput screening of synthetic chemical libraries against many potential therapeutic targets, including new targets identified from the human genome sequencing project, largely to the exclusion of natural products, and discovery rates dropped dramatically. Nonetheless, natural products continued to provide key scaffolds for drug development. In the current millennium, it was discovered from genome sequencing that microbes with large genomes have the capacity to produce about ten times as many secondary metabolites as was previously recognized. Indeed, the most gifted actinomycetes have the capacity to produce around 30-50 secondary metabolites. With the precipitous drop in cost for genome sequencing, it is now feasible to sequence thousands of actinomycete genomes to identify the "biosynthetic dark matter" as sources for the discovery of new and novel secondary metabolites. Advances in bioinformatics, mass spectrometry, proteomics, transcriptomics, metabolomics and gene expression are driving the new field of microbial genome mining for applications in natural product discovery and development.

Substrate Activity Screening (SAS) and Related Approaches in Medicinal Chemistry.

Substrate activity screening (SAS) was presented a decade ago by Ellman and co-workers as a straightforward methodology for the identification of fragment-sized building blocks for enzyme inhibitors. Ever since, SAS and variations derived from it have been successfully applied to the discovery of inhibitors of various families of enzymatically active drug targets. This review covers key achievements and challenges of SAS and related methodologies, including the modified substrate activity screening (MSAS) approach. Special attention is given to the kinetic and thermodynamic aspects of these methodologies, as a thorough understanding thereof is crucial for successfully transforming the identified fragment-sized hits into potent inhibitors.