The Irreversible FGFR Inhibitor KIN-3248 Overcomes FGFR2 Kinase Domain Mutations.

PURPOSE: FGFR2 and FGFR3 show oncogenic activation in many cancer types, often through chromosomal fusion or extracellular domain mutation. FGFR2 and FGFR3 alterations are most prevalent in intrahepatic cholangiocarcinoma (ICC) and bladder cancers, respectively, and multiple selective reversible and covalent pan-FGFR tyrosine kinase inhibitors (TKI) have been approved in these contexts. However, resistance, often due to acquired secondary mutations in the FGFR2/3 kinase domain, limits efficacy. Resistance is typically polyclonal, involving a spectrum of different mutations that most frequently affect the molecular brake and gatekeeper residues (N550 and V565 in FGFR2). EXPERIMENTAL DESIGN: Here, we characterize the activity of the next-generation covalent FGFR inhibitor, KIN-3248, in preclinical models of FGFR2 fusion+ ICC harboring a series of secondary kinase domain mutations, in vitro and in vivo. We also test select FGFR3 alleles in bladder cancer models. RESULTS: KIN-3248 exhibits potent selectivity for FGFR1-3 and retains activity against various FGFR2 kinase domain mutations, in addition to being effective against FGFR3 V555M and N540K mutations. Notably, KIN-3248 activity extends to the FGFR2 V565F gatekeeper mutation, which causes profound resistance to currently approved FGFR inhibitors. Combination treatment with EGFR or MEK inhibitors potentiates KIN-3248 efficacy in vivo, including in models harboring FGFR2 kinase domain mutations. CONCLUSIONS: Thus, KIN-3248 is a novel FGFR1-4 inhibitor whose distinct activity profile against FGFR kinase domain mutations highlights its potential for the treatment of ICC and other FGFR-driven cancers.

Discovery of KIN-3248, An Irreversible, Next Generation FGFR Inhibitor for the Treatment of Advanced Tumors Harboring FGFR2 and/or FGFR3 Gene Alterations.

Fibroblast growth factor receptor (FGFR) alterations are present as oncogenic drivers and bypass mechanisms in many forms of cancer. These alterations can include fusions, amplifications, rearrangements, and mutations. Acquired drug resistance to current FGFR inhibitors often results in disease progression and unfavorable outcomes for patients. Genomic profiling of tumors refractory to current FGFR inhibitors in the clinic has revealed several acquired driver alterations that could be the target of next generation therapeutics. Herein, we describe how structure-based drug design (SBDD) was used to enable the discovery of the potent and kinome selective pan-FGFR inhibitor KIN-3248, which is active against many acquired resistance mutations. KIN-3248 is currently in phase I clinical development for the treatment of advanced tumors harboring FGFR2 and/or FGFR3 gene alterations.

Rational discovery of a highly novel and selective mTOR inhibitor.

Aided by Structure Based Drug Discovery (SBDD), we rapidly designed a highly novel and selective series of mTOR inhibitors. This chemotype conveys exquisite kinase selectivity, excellent in vitro and in vivo potencies and ADME safety profiles. These compounds could serve as good tools to explore the potential of TORC inhibition in various human diseases.

Disarming Pseudomonas aeruginosa virulence factor LasB by leveraging a Caenorhabditis elegans infection model.

The emergence of antibiotic resistance places a sense of urgency on the development of alternative antibacterial strategies, of which targeting virulence factors has been regarded as a "second generation" antibiotic approach. In the case of Pseudomonas aeruginosa infections, a proteolytic virulence factor, LasB, is one such target. Unfortunately, we and others have not been successful in translating in vitro potency of LasB inhibitors to in vivo efficacy in an animal model. To overcome this obstacle, we now integrate in silico and in vitro identification of the mercaptoacetamide motif as an effective class of LasB inhibitors with full in vivo characterization of mercaptoacetamide prodrugs using Caenorhabditis elegans. We show that one of our mercaptoacetamide prodrugs has a good selectivity profile and high in vivo efficacy, and confirm that LasB is a promising target for the treatment of bacterial infections. In addition, our work highlights that the C. elegans infection model is a user-friendly and cost-effective translational tool for the development of anti-virulence compounds.

Synthesis/biological evaluation of hydroxamic acids and their prodrugs as inhibitors for Botulinum neurotoxin A light chain.

Botulinum neurotoxin A (BoNT/A) is the most potent toxin known. Unfortunately, it is also a potential bioweapon in terrorism, which is without an approved therapeutic treatment once cellular intoxication takes place. Previously, we reported how hydroxamic acid prodrug carbamates increased cellular uptake, which translated to successful inhibition of this neurotoxin. Building upon this research, we detail BoNT/A protease molecular modeling studies accompanied by the construction of small library of hydroxamic acids based on 2,4-dichlorocinnamic hydroxamic acid scaffold and their carbamate prodrug derivatization along with the evaluation of these molecules in both enzymatic and cellular models.

The C-terminus of Botulinum A Protease Has Profound and Unanticipated Kinetic Consequences Upon the Catalytic Cleft.

Botulinum neurotoxins (BoNTs) are among the most deadly poisons known though ironically, they also are of great therapeutic utility. A number of research programs have been initiated to discover small molecule inhibitors of BoNTs metalloprotease activity. Many, though not all of these programs have screened against a truncated and more stable form of the enzyme, that possess comparable catalytic properties to the full length enzyme. Interestingly, several classes of inhibitors notably the hydroxamates, display a large shift in potency between the two enzyme forms. In this report we compare the kinetics of active-site, alpha-exosite and beta-exosite inhibitors versus truncated and full length enzyme. Molecular dynamics simulations conducted with the truncated and homology models of the fully length BoNT LC/A indicate the flexibility of the C-terminus of the full length enzyme is responsible for the potency shifts of active-site proximally binding inhibitors while distal binding (alpha-exosite) inhibitors remain equipotent.

Building a three-dimensional model of CYP2C9 inhibition using the Autocorrelator: an autonomous model generator.

In modern day drug discovery campaigns, computational chemists have to be concerned not only about improving the potency of molecules but also reducing any off-target ADMET activity. There are a plethora of antitargets that computational chemists may have to consider. Fortunately many antitargets have crystal structures deposited in the PDB. These structures are immediately useful to our Autocorrelator: an automated model generator that optimizes variables for building computational models. This paper describes the use of the Autocorrelator to construct high quality docking models for cytochrome P450 2C9 (CYP2C9) from two publicly available crystal structures. Both models result in strong correlation coefficients (R² > 0.66) between the predicted and experimental determined log(IC50) values. Results from the two models overlap well with each other, converging on the same scoring function, deprotonated charge state, and predicted the binding orientation for our collection of molecules.

Synthesis and molecular modeling provide insight into a Pseudomonas aeruginosa quorum sensing conundrum.

The triphenyl amide/ester 12 was originally reported to be a potent mimic of the natural 3-oxo-dodecanoyl homoserine lactone quorum sensing molecule in Pseudomonas aeruginosa. However, explicit synthesis/chemical characterization was lacking, and a later report providing protein crystallographic data inferred 12 to be incorrect, with 9 now being the surmised structure. Because of these inconsistencies and our interest in quorum sensing molecules utilized by gram-negative bacteria, we found it necessary to synthesize 9 and 12 to test for agonistic activity in a P. aeruginosa reporter assay. Despite distinct regiochemical differences, both 9 and 12 were found to have comparable EC(50) values. To reconcile these unanticipated findings, modeling studies were conducted, and both compounds were revealed to have comparable properties for binding to the LasR receptor.

Illuminator: increasing synergies between medicinal and computational chemists.

We present Illuminator, a user-friendly web front end to computational models such as docking and 3D shape similarity calculations. Illuminator was specifically created to allow non-experts to design and submit molecules to computational chemistry programs. As such it provides a simple user interface allowing users to submit jobs starting from a 2D structure. The models provided are pre-optimized by computational chemists for each specific target. We provide an example of how Illuminator was used to prioritize the design of molecular substituents in the Anadys HCV Polymerase (NS5B) project. With 7500 submitted jobs in 1.5 years, Illuminator has allowed project teams at Anadys to accelerate the optimization of novel leads. It has also improved communication between project members and increased demand for computational drug discovery tools.

5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase.

The discovery of 5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC50 <0.10 microM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.

4-(1,1-Dioxo-1,4-dihydro-1lambda6-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-ones as potent inhibitors of HCV NS5B polymerase.

4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).

Pyrrolo[1,2-b]pyridazin-2-ones as potent inhibitors of HCV NS5B polymerase.

Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda6-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones: Part 4. Optimization of DMPK properties.

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as potent inhibitors of genotype 1 HCV NS5B polymerase focusing on the optimization of their drug metabolism and pharmacokinetics (DMPK) profiles. This investigation led to the discovery of potent inhibitors with improved DMPK properties.

Hydra: a self regenerating high performance computing grid for drug discovery.

Computer aided drug design is progressing and playing an increasingly important role in drug discovery. Computational methods are being used to evaluate larger and larger numbers of real and virtual compounds. New methods based on molecular simulations that take protein and ligand flexibility into account also contribute to an ever increasing need for compute time. Computational grids are therefore becoming a critically important tool for modern drug discovery, but can be expensive to deploy and maintain. Here, we describe the low cost implementation of a 165 node, computational grid at Anadys Pharmaceuticals. The grid makes use of the excess computing capacity of desktop computers deployed throughout the company and of outdated desktop computers which populate a central computing grid. The performance of the grid grows automatically with the size of the company and with advances in computer technology. To ensure the uniformity of the nodes in the grid, all computers are running the Linux operating system. The desktop computers run Linux inside MS Windows using coLinux as virtualization software. HYDRA has been used to optimize computational models, for virtual screening and for lead optimization.