A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

Tuning the Reactivity of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators for Optimal in Vivo Efficacy.

Dimethyl fumarate 1 is approved for the treatment of multiple sclerosis but is also associated with off-target activation of the niacin receptor. By using a tetrazolone or triazolone bioisostere approach to the fumarate and vinyl sulfone series of Nrf2 activators, we have optimized the electrophilicity of the double bond to tune the on-target Nrf2 activation with PK properties to achieve efficacy in animal models of multiple sclerosis. The study linked highly potent, highly electrophilic molecules to low plasma stability and, subsequently, limited efficacy. By contrast, a sulfonylvinyltriazolone 17 retains on-target potency but shows much weaker electrophilic potential. As a consequence, in vivo high exposures of 17 are obtained, resulting in efficacy in the EAE model similar to that observed for DMF. 17 (R079) is Ames negative, is not cytotoxic to cells, and shows little inhibition of either the niacin receptor or a panel of off-target receptors.

Development of a MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although cSCC contributes to substantial morbidity and mortality in high-risk individuals, deployment of otherwise effective chemoprevention of cSCC is limited by toxicities. Our systematic computational drug repurposing screen predicted that selumetinib, a MAPK (mitogen-activated protein kinase) kinase inhibitor (MEKi), would reverse transcriptional signatures associated with cSCC development, consistent with our genomic analysis implicating MEK as a chemoprevention target. Although systemic MEKi suppresses the formation of cSCC in mice, systemic MEKi can cause severe adverse effects. Here, we report the development of a metabolically labile MEKi, NFX-179, designed to potently and selectively suppress the MAPK pathway in the skin before rapid metabolism in the systemic circulation. NFX-179 was identified on the basis of its biochemical and cellular potency, selectivity, and rapid metabolism upon systemic absorption. In our ultraviolet-induced cSCC mouse model, topical application of NFX-179 gel reduced the formation of new cSCCs by an average of 60% at doses of 0.1% and greater at 28 days. We further confirmed the localized nature of these effects in an additional split-mouse randomized controlled study where suppression of cSCC was observed only in drug-treated areas. No toxicities were observed. NFX-179 inhibits the growth of human SCC cell lines in a dose-dependent manner, and topical NFX-179 application penetrates human skin and inhibits MAPK signaling in human cSCC explants. Together, our data provide a compelling rationale for using topical MEK inhibition through the application of NFX-179 gel as an effective strategy for cSCC chemoprevention.

Early Drug Discovery and Development of Novel Cancer Therapeutics Targeting DNA Polymerase Eta (POLH).

Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme's ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.

Fragment- and structure-based drug discovery for developing therapeutic agents targeting the DNA Damage Response.

Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with ∼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of ∼2-8% and crystal structures from ∼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).

A Single-Step Synthesis of Azetidine-3-amines.

The azetidine group is frequently encountered within contemporary medicinal chemistry. However, the introduction of an azetidine can be synthetically challenging. Herein, a straightforward synthesis of azetidine-3-amines, starting from a bench stable, commercial material is presented. The reaction tolerates common functionality and proceeds in moderate-to-high yield with secondary amines, and moderate-to-low yield with primary amines. The methodology compares favorably to alternative procedures and can be utilized in "any-stage" functionalization, including late-stage azetidinylation of approved drugs and other compounds with pharmacological activity.

Toward Orally Absorbed Prodrugs of the Antibiotic Aztreonam. Design of Novel Prodrugs of Sulfate Containing Drugs. Part 2.

Aztreonam, first discovered in 1980, is an FDA approved, intravenous, monocyclic beta-lactam antibiotic. Aztreonam is active against Gram-negative bacteria and is still used today. The oral bioavailability of aztreonam in humans is less than 1%. Herein we describe the design and synthesis of potential oral prodrugs of aztreonam.

Orally Absorbed Derivatives of the ß-Lactamase Inhibitor Avibactam. Design of Novel Prodrugs of Sulfate Containing Drugs.

Only one FDA-approved ß-lactamase inhibitor has ever been orally available: clavulanic acid, approved in 1984. Avibactam, approved by FDA in 2015, is the first of a new class of BLIs called diazabicyclooctanes, or "DBOs". This class has much broader coverage than clavulanic acid but can only be administered by intravenous injection. Herein, we describe the synthesis and testing of the first approved BLI to be rendered orally bioavailable since clavulanic acid (1984).

Enantioselective synthesis of an octahydroindolizine (indolizidine) alcohol using an enzymatic resolution.

A homo-chiral synthesis of (7R,8aS)-octahydro-5,5-dimethylindolizin-7-amine 8 and (7S, 8aS)-octahydro-5,5-dimethylindolizin-7-ol 9, amine building blocks which have found applications within the pharmaceutical industry, is presented. The approach uses a Novozym 435-mediated kinetic resolution of racemic octahydroindolizine (indolizidine) alcohol 13 as a key step (up to 100 g scale).

A one-pot synthesis of tetrazolones from acid chlorides: understanding functional group compatibility, and application to the late-stage functionalization of marketed drugs.

A one-pot and scalable synthesis of tetrazolones (tetrazol-5-ones) from acid chlorides using azidotrimethylsilane is presented. The reaction tolerates many functional groups and can furnish aryl-, heteroaryl-, alkenyl-, or alkyl-substituted tetrazolone products in moderate to excellent yield (14-94%). No reduction in yield was observed when the reaction was undertaken on a larger-scale (20-36 g). The method could be used for the late-stage functionalization of pharmaceuticals, to provide tetrazolone congeners of the marketed drugs aspirin, indomethacin, probenecid, telmisartan, bexarotene, niacin (vitamin B3), and the active metabolite of the recently-launched immuno-modulatory agent, BG-12 (Tecfidera®). The ability of a tetrazolone group to serve as a bioisostere of a carboxylic acid, and to improve drug pharmacokinetic profiles is also highlighted.

Tetrazolone as an acid bioisostere: application to marketed drugs containing a carboxylic acid.

Matched molecular pair analysis was used to evaluate the ability of a tetrazolone group to act as a bioisostere of a carboxylic acid. Compound 7, a tetrazolone of the anti-hypertensive drug, telmisartan 6, was shown to be a potent AT1 antagonist (Kb = 0.14 nM), with activity comparable to telmisartan itself (Kb = 0.44 nM). Additionally, compound 9, a tetrazolone congener of the marketed anti-cancer agent, bexarotene 8, was shown to be an agonist at the retinoid X receptor alpha (EC50 = 64 nM). Compounds containing a tetrazolone group showed similar microsomal stability and plasma protein binding to marketed acid counterparts, while also reducing the value for clog P. Furthermore, compound 7 displayed an improved rat pharmacokinetic profile cf. telmisartan 6. Taken together, the results demonstrate that a tetrazolone group may serve as a bioisostere for a carboxylic acid.

Identification of orally-bioavailable antagonists of the TRPV4 ion-channel.

Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited optimization program. The leading compounds obtained from this exercise, RN-1665 23 and RN-9893 26, showed moderate oral bioavailability when dosed to rats. The lead molecule, RN-9893 26, inhibited human, rat and murine variants of TRPV4, and showed excellent selectivity over related TRP receptors, such as TRPV1, TRPV3 and TRPM8. The overall profile for RN-9893 may permit its use as a proof-of-concept probe for in vivo applications.

Synthesis of trans-2-(trifluoromethyl)cyclopropanes via Suzuki reactions with an N-methyliminodiacetic acid boronate.

trans-2-(Trifluoromethyl)cyclopropylboronic acid N-methyliminodiacetic acid (MIDA) ester 5 was synthesized as a pure diastereomer from vinylboronic acid MIDA ester and (trifluoromethyl)diazomethane in a single step. An X-ray study confirmed the trans-stereochemistry around the cyclopropyl ring. Use of 5 in Suzuki reactions, with a variety of aryl or heteroaryl coupling partners, provided trans-2-(trifluoromethyl)cyclopropyl products in moderate to excellent yields (17-90%).

A concise one-pot synthesis of trifluoromethyl-containing 2,6-disubstituted 5,6,7,8-tetrahydroquinolines and 5,6,7,8-tetrahydronaphthyridines.

5,6,7,8-Tetrahydroquinolines and 5,6,7,8-tetrahydronaphthyridines with appended trifluoromethyl groups are valuable chemotypes in medicinal chemistry due to the presence of a partially-saturated bicyclic ring and metabolically-stable CF(3) group. (1)H NMR studies were used to optimize the preparation of such compounds, using a three-step/one-pot procedure, to provide novel 2,6-disubstitued derivatives with a tertiary-substituent. Racemic 2,6-disubstituted tetrahydroquinolines were separated by chiral HPLC to provide single enantiomers.

TRPM8 biology and medicinal chemistry.

TRPM8 belongs to the TRPM Melastatin subfamily of Transient Receptor Potential (TRP) ion channels. Activated by cool temperatures and mimetic ligands, such as menthol and icilin, TRPM8 has been shown to play a role in thermoreception and is expressed in peripheral nerves. TRPM8 is also expressed in other tissues which are not exposed to temperature fluctuations, such as the prostate. The recent advancement of a TRPM8 agonist into the clinic for the treatment of prostate cancer suggests that the channel plays a role in some human pathologies. As more drug-like and selective agonists and antagonists of TRPM8 become available, in vivo pharmacology studies will complement already published knockout data to further our understanding of the role of TRPM8 in human disease.

TRPV4 agonists and antagonists.

TRPV4 belongs to the TRPV subfamily of Transient Receptor Potential (TRP) ion channels. This year marks the 10 year anniversary of the discovery of this polymodal ion channel which is activated by a variety of stimuli including warm temperatures, hypotonicity and endogenous lipids. Coupled with a widespread tissue distribution, this activation profile has resulted in a large number of disparate physiological functions for TRPV4. These range from temperature monitoring in skin keratinocytes to osmolarity sensing in kidneys, sheer stress detection in blood vessels and osteoclast differentiation control in bone. As knowledge of its physiological roles has expanded, interest in targeting TRPV4 modulation for therapeutic purposes has arisen and is now focused on several areas. First, as with related TRP channels TRPV1, TRPV3, TRPM8 and TRPA1, TRPV4 antagonism is being considered for inflammatory and neuropathic pain treatment. Recent work conducted using KO mice and agonists 4αPDD and GSK1016790A suggests bladder dysfunctions may also be targeted. Additionally, ventilator-induced lung injury has emerged as another potential indication for TRPV4 antagonists. Herein we review the known small molecule modulators of TRPV4 and relate progress made in identifying potent, selective and bioavailable agonists and antagonists to interrogate this ion channel in vivo.

5-benzyloxytryptamine as an antagonist of TRPM8.

5-Benzyloxytryptamine 19 was found to act as an antagonist of the TRPM8 ion-channel. For example, 19 had an IC(50) of 0.34 µM when menthol was used as the stimulating agonist. Related commercially-available tryptamine derivatives showed diminished, or no antagonist activity at TRPM8. The structural similarity of 5-benzyloxytryptamine to other literature TRPM8 antagonists was noted.

Oxime derivatives related to AP18: Agonists and antagonists of the TRPA1 receptor.

AP18 1 was recently disclosed as an antagonist of the TRPA1 receptor by the research group of Patapoutian. However, no detailed structure-activity relationships around 1 have been disclosed. Thus, a small number of oximes related to AP18 were examined in order to characterize the determinants of TRPA1 activity. Congeners of AP18 were found to possess both agonist and antagonist activity, suggesting that AP18 may behave as a covalent antagonist of the TRPA1 ion-channel.

Mining biologically-active molecules for inhibitors of fatty acid amide hydrolase (FAAH): identification of phenmedipham and amperozide as FAAH inhibitors.

The screening of known medicinal agents against new biological targets has been shown to be a valuable approach for revealing new pharmacology of marketed compounds. Recently, carbamate, urea and ketone inhibitors of fatty acid amide hydrolase (FAAH) have been described as promising treatments for pain, anxiety, depression and other CNS-related conditions. In order to find novel FAAH inhibitors, a focused screen of molecules containing potentially reactive moieties or having in vivo effects that are possibly relevant to the biology of FAAH was conducted. These studies revealed phenmedipham 13 and amperozide 14 to be inhibitors of human FAAH, with an IC(50) of 377 nM and 1.34 microM, respectively.

Identification and characterization of novel TRPV4 modulators.

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies.