Fragment-based approaches in drug discovery and chemical biology

Fragment-based approaches to finding novel small molecules that bind to proteins are now firmly established in drug discovery and chemical biology. Initially developed primarily in a few centers in the biotech and pharma industry, this methodology has now been adopted widely in both the pharmaceutical industry and academia. After the initial success with kinase targets, the versatility of this approach has now expanded to a broad range of different protein classes. Herein we describe recent fragment-based approaches to a wide range of target types, including Hsp90, β-secretase, and allosteric sites in human immunodeficiency virus protease and fanesyl pyrophosphate synthase. The role of fragment-based approaches in an academic research environment is also examined with an emphasis on neglected diseases such as tuberculosis. The development of a fragment library, the fragment screening process, and the subsequent fragment hit elaboration will be discussed using examples from the literature.     

Fragment-based discovery of a potent NAMPT inhibitor

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.     

Fragment-based discovery of JAK-2 inhibitors

Fragment-based hit identification coupled with crystallographically enabled structure-based drug design was used to design potent inhibitors of JAK-2. After two iterations from fragment 1, we were able to increase potency by greater than 500-fold to provide sulfonamide 13, a 78-nM JAK-2 inhibitor.     

Fragment-based drug discovery using NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience.     

Fragment-based ligand discovery meets phage display.

In a powerful complement to traditional ligand discovery methods such as high-throughput screening, fragment-based ligand discovery methods identify ligands piece by piece. A recent advance combines the concepts of fragment-based ligand discovery with phage-display technology to yield bivalent kinase inhibitors with high potency and specificity.     

Fragment-based discovery of focal adhesion kinase inhibitors

Chemically diverse fragment hits of focal adhesion kinase (FAK) were discovered by surface plasmon resonance (SPR) screening of our in-house fragment library. Site specific binding of the primary hits was confirmed in a competition setup using a high-affinity ATP-site inhibitor of FAK. Protein crystallography revealed the binding mode of 41 out of 48 selected fragment hits within the ATP-site. Structural comparison of the fragment binding modes with a DFG-out inhibitor of FAK initiated first synthetic follow-up optimization leading to improved binding affinity.     

Fragment-based discovery and optimization of BACE1 inhibitors

A novel series of 2-aminobenzimidazole inhibitors of BACE1 has been discovered using fragment-based drug discovery (FBDD) techniques. The rapid optimization of these inhibitors using structure-guided medicinal chemistry is discussed.     

Progress in maize gene discovery: a project update

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon (RescueMu). Current achievements of the MGDP (NSF 98–72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor . In addition, ZmDB contains links to other plant and genetics websites. Electronic Publication     

Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein

Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts.     

Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.     

Teratogen update: lead and pregnancy

This review focuses on the impacts of lead exposure on reproductive health and outcomes. High levels of paternal lead exposure (>40 microg/dl or >25 microg/dl for a period of years) appear to reduce fertility and to increase the risks of spontaneous abortion and reduced fetal growth (preterm delivery, low birth weight). Maternal blood lead levels of approximately 10 microg/dl have been linked to increased risks of pregnancy hypertension, spontaneous abortion, and reduced offspring neurobehavioral development. Somewhat higher maternal lead levels have been linked to reduced fetal growth. Some studies suggest a link between increased parental lead exposure and congenital malformations, although considerable uncertainty remains regarding the specific malformations and the dose-response relationships. Common methodological weaknesses of studies include potential exposure misclassifications due to the frequent unavailability of exposure biomarker measurements at biologically appropriate times and uncertainty regarding the best exposure biomarker(s) for the various outcomes. A special concern with regard to the pregnant woman is the possibility that a fetus might be exposed to lead mobilized from bone stores as a result of pregnancy-related metabolic changes, making fetal lead exposure the result of exposure to exogenous lead during pregnancy and exposure to endogenous lead accumulated by the woman prior to pregnancy. By reducing bone resorption, increased calcium intake during the second half of pregnancy might reduce the mobilization of lead from bone compartments, even at low blood lead levels. Subgroups of women who incurred substantial exposures to lead prior to pregnancy should be considered to be at increased risk.     

Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with 1–10 mM binding affinity (K_D) were iteratively optimized to give leads with 200 nM biochemical activity and low μM cellular activity in a Replicon assay.     

Fragment-based discovery of hydroxy-indazole-carboxamides as novel small molecule inhibitors of Hsp90

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe the identification of novel small molecular weight inhibitors of Hsp90 using a fragment based approach. Fragments were selected by docking, tested in a biochemical assay and the confirmed hits were crystallized. Information gained from X-ray structures of these fragments and other chemotypes was used to drive the fragment evolution process. Optimization of these high μM binders resulted in 3-benzylindazole derivatives with significantly improved affinity and anti-proliferative effects in different human cancer cell lines.     

Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

The development of low μM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K_i = 1.4 ± 0.3 μM) was found to be selective when tested against a panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA, and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure–activity relationships observed in vitro and revealed further possibilities for compound development.     

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.     

GPCRs: an update on structural approaches to drug discovery

G-protein-coupled receptors (GPCRs) are a major opportunity for drug discovery in the post-genomic era. There are thought to be more than 500 therapeutically relevant GPCRs out of a total of over 700 identified to date, although only one, rhodopsin, has been the subject of a full 3D X-ray crystallography study. Two structurally related proteins, bacteriorhodopsin and sensory rhodopsin, which are not GPCRs but are part of the seven-helix membrane receptor family, have also been the subject of X-ray crystallographic studies and have been used in GPCR modeling studies. The significant differences between these rhodopsin structures, the relatively low sequence homology between individual GPCRs, and some difficulties in rationalizing point-mutation data suggests that homology-based molecular modeling alone will not provide the accurate structural information on individual receptors required for ligand design and in silico screening. In the absence of such structural information, several approaches can be used to assist in the discovery of ligands.     

Novel strategies in antifungal lead discovery

There have been significant developments in fungal genomics over the past year. The recently released genome sequences of Aspergillus fumigatus and Cryptococcus neoformans have provided unprecedented opportunities for comparative genomics studies of many clinically relevant fungal pathogens. Emerging experimental analysis tools, such as fitness profiling and protein microarrays, have greatly enhanced our ability to conduct genome-wide functional studies.