Corey Strickland

Discovery of Cyclic Acylguanidines as Highly Potent and Selective beta-Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I-Inhibitor Design and Validation

A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

Application of Fragment-Based NMR Screening, X-ray Crystallography, Structure-Based Design, and Focused Chemical Library Design to Identify Novel μM Leads for the Development of nM BACE-1 (β-Site APP Cleaving Enzyme 1) Inhibitors

Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper

Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

Background: Bezlotoxumab is a neutralizing antibody targeting toxin B of Clostridium difficile. Results: The structure of bezlotoxumab bound to a fragment of toxin B reveals its epitopes and mechanism of neutralization. Conclusion: The epitopes overlap with two of the presumed carbohydrate binding pockets, preventing binding of the toxin to target host cells. Significance: The data provide a molecular basis for neutralization by this clinically important antibody. The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Inhibitors of BACE for treating Alzheimer's disease: a fragment-based drug discovery story

Several fragment-based methods have been applied to the discovery of new lead sources for inhibitors of BACE1, an important therapeutic target for Alzheimers disease. Among the most common fragment hits were various amidine-containing molecules in which the amidine engaged in discrete H-bond donor-acceptor interaction with the BACE1 catalytic dyad. Structure and medicinal chemistry knowledge-based optimization with emphasis on ligand efficiency resulted in identification of a key pharmacophore comprising a non-planar cyclic amidine scaffold directly attached to a phenyl group projecting into S1. This key pharmacophore is a common feature of known clinical candidates and has dominated the recent patent literature. A structural comparison of the non-planar cyclic amidine motif with other BACE1 pharmacophores highlights its uniqueness and distinct advantages.

Cloning, heterologous expression, and distinct substrate specificity of protein farnesyltransferase from Trypanosoma brucei.

Protein prenylation occurs in the protozoan that causes African sleeping sickness (Trypanosoma brucei), and the protein farnesyltransferase appears to be a good target for developing drugs. We have cloned the α- and β-subunits of T. brucei protein farnesyltransferase (TB-PFT) using nucleic acid probes designed from partial amino acid sequences obtained from the enzyme purified from insect stage parasites. TB-PFT is expressed in both bloodstream and insect stage parasites. Enzymatically active TB-PFT was produced by heterologous expression in Escherichia coli. Compared with mammalian protein farnesyltransferases, TB-PFT contains a number of inserts of >25 residues in both subunits that reside on the surface of the enzyme in turns linking adjacent α-helices. Substrate specificity studies with a series of 20 peptides SSCALX (where X indicates a naturally occurring amino acid) show that the recombinant enzyme behaves identically to the native enzyme and displays distinct specificity compared with mammalian protein farnesyltransferase. TB-PFT prefers Gln and Met at theX position but not Ser, Thr, or Cys, which are good substrates for mammalian protein farnesyltransferase. A structural homology model of the active site of TB-PFT provides a basis for understanding structure-activity relations among substrates and CAAX mimetic inhibitors.

Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab

Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility.

Combining NMR and X-ray Crystallography in Fragment-Based Drug Discovery: Discovery of Highly Potent and Selective BACE-1 Inhibitors

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimers disease in a clinical setting.

Efficacy, Pharmacokinetics, and Metabolism of Tetrahydroquinoline Inhibitors of Plasmodium falciparum Protein Farnesyltransferase

ABSTRACT New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Zn-binding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.

Trihalobenzocycloheptapyridine analogues of Sch 66336 as potent inhibitors of farnesyl protein transferase.

SCH 66336 is a trihalo tricyclic compound that is currently undergoing Phase II clinical trials for the treatment of solid tumors. Modifications of SCH 66336 by incorporating such groups as amides, acids, esters, ureas and lactams off the first or the distal piperidine (from the tricycle) provided potent FPT inhibitors some of which exhibited good cellular activity. A number of these compounds incorporate properties that might improve pharmacokinetic stability of these inhibitors by virtue of their increased solubility or by their change in log P.

Discovery of potent iminoheterocycle BACE1 inhibitors.

The synthesis of a series of iminoheterocycles and their structure-activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aβ40 levels upon subcutaneous and oral administration to rats.