Covalent Modifiers: An Orthogonal Approach to Drug Design - Journal

 Copyright 2009 by the American Chemical Society

Volume 52, Number 5

March 12, 2009

PerspectiVe Covalent Modifiers: An Orthogonal Approach to Drug Design Michele H. Potashman* and Mark E. Duggan*,§ Department of Medicinal Chemistry, Amgen Inc., One Kendall Square, Building 1000, Cambridge, Massachusetts 02139 ReceiVed July 11, 2008

Introduction In an effort to discover small molecule drug candidates with attractive toxicity profiles, the pharmaceutical industry has predominantly sought compounds that modulate target proteins through noncovalent interactions. Therefore, high throughput screening strategies and screening collections have been tailored to identify leads that display this binding profile. Scientists will often disregard or filter out screening hits containing potentially reactive functionality or compounds that exhibit noncompetitive kinetics (due to either covalent or noncovalent interactions). This is not surprising, as it is well-known that indiscriminant reactivity can result in covalent binding to proteins, DNA, and glutathione (GSHa), which can herald unfavorable toxicological outcomes.1 These toxicological events may occur either acutely or as a delayed response due to either immunological or histological factors (Table 1).2 In the case of immunological (allergic) response, either a drug-protein complex or a degradation product of such a complex can act as a stimulant for the immune system (Figure 1). Even when compounds present no grave toxicological outcome in preclinical models, idiosyncratic reactions can be manifested in human clinical trials or arise once the entity is exposed to a larger patient pool. Unfortunately, predicting life-threatening idiosyncratic adverse events in humans at the preclinical stage has been evasive. As a result, * To whom correspondence should be addressed. For M.H.P.: phone, (617) 444-5010; fax, (617) 577-9288; e-mail, [email protected]. For M.E.D.: phone, (617) 583-1983; e-mail, [email protected]. § Current address: Link Medicine Inc., 161 First Street, Cambridge, MA 02142. a Abbreviations: GSH, glutathione; AChE, acetylcholinesterase; PLP, pyridoxal 5′-phosphate; GABA-AT, γ-aminobutyric acid aminotransferase; PPIs, proton pump inhibitors; ID-I, type 1 thyroxine 5′-deiodinase; (HCV)NS3 · 4A, hepatitis C virus NS3 serine protease, EGFR or erB1, epidermal growth factor receptor; HER-2 or erB1, human epidermal growth factor receptor 2; MAO-B, monoamine oxidase B; DPP-IV, dipeptidyl peptidase; GLP-1, glucagon-like peptide 1; Cat K, cathepsin K.

there is often resistance toward the development of drugs containing modestly reactive functionality, even when the reactivity is confined to the biochemical target. It is widely acknowledged that nonspecific covalent binding should be avoided or minimized when optimizing drug properties. In fact, the industry now monitors for levels of metabolic activation at the preclinical stage to eliminate potential development candidates that furnish undesirable levels of drug-protein complexes as a strategy to reduce the potential for organ toxicity or idiosyncratic reactions.1a There are examples where covalent drug (or metabolite)-protein adducts do not lead to toxicity, such as is observed with 3′-hydroxyacetanilide, the meta-isomer of acetaminophen.3 However, since it is difficult to predict physiological responses to a drug-protein adduct, nonspecific covalent binding is undesired. Alternatively, there are instances where controlled, targetspecific covalent modification has proven useful. In most instances this has not been the strategy but rather discovered in hindsight. In ideal cases the covalent modifier is typically poorly reactive with solution nucleophiles under physiological conditions but yet upon appropriate positioning will selectively react with a nucleophile within the target protein.4 Herein, we review a variety of examples in which therapeutic targets are covalently bound by small-molecule drugs or by compounds in advanced clinical development. The covalent interactions can be either reversible or irreversible, depending on the reaction partners. The drugs within this review contain functionality ranging from potentially reactive with solution nucleophiles, as in the case of an epoxide or activated ester, to less-reactive functionality that react upon target binding, such as carbamates and nitriles. Where known, we include the proposed mechanisms of inhibition, crystal structures, and models illustrating the binding mode, as well as biochemical proof of covalent inhibition and

10.1021/jm8008597 CCC: $40.75  2009 American Chemical Society Published on Web 02/09/2009

1232 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5

Figure 1. Postulated mechanism for drug hypersensitivity reactions. This pathway can be achieved by either a reactive parent compound or a metabolite.

information on the reversibility of binding. Finally, in the Perspective section we suggest medicinal chemistry strategies that enable rational approaches to therapeutics that bind through targeted covalent interactions. Methods and Materials Method of Analysis. This review article focuses on small molecule drugs and late stage clinical candidates that modify their target protein through a covalent interaction. The targets and compounds presented herein are restricted to those that have published biochemical proof of covalent target modulation. Alternatively, when crystal structures and proposed binding models are convincing of covalent inhibition, those examples are included as well. Proposed or studied chemical mechanisms have been included when documented. The reversibility of the covalent adduction is mentioned, if known. In this discussion, reversibility refers to the event where drug dissociates from the protein as the parent form or as a modified derivative. If there is no mechanism for the removal of the covalent drug-derived moiety or if that removal is not detected, the process is classified as irreversible. However, compounds with slow off-rates due to exclusively noncovalent interactions (such as strong van der Waals interactions, strong metal chelators,5 or irreversible reorganization of the protein upon substrate binding6) have not been included in this discussion. Although noncovalent binders that exhibit slow off-rates may display overall pharmacodynamic properties commensurate with a covalent inhibitor, these compounds have been excluded from this review. In addition to covalent adduction by the parent compound, several examples of covalent modification by a major metabolite are presented. In these cases, the parent compound prodrug7 is inactive and the observed pharmacodynamic activity results from a defined metabolite. Scope. As there exists no comprehensive source systematically detailing the chemical mechanism of action for marketed drugs or any formalized means of classifying drug mechanism at the target level, the data presented in this review have been gathered from several sources. The database and literature searches conducted were broad in scope; however, it is possible that some drugs may have been overlooked. Marketed or investigational drugs that bind the therapeutic target covalently were identified by searching the following major sources: (a) electronic databases such as the Food and Drug Administration (FDA) drug labels, European Medicines Evaluation Agency (EMEA) drug labels, Scifinder (CAS database), IDdb (Investigational Drugs database), Physicians Desk Reference (PDR); (b) published litera-

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ture (including several review articles)8 and presentations at national chemistry meetings, such as the American Chemical Society meetings. Because of the lack of complete information on the total number of drug targets for which therapeutic agents exist or a comprehensive list of targets that form covalently bound drug adducts, no attempt was made to analyze the frequency or advantage of covalent inhibition with respect to noncovalent inhibition. Additionally, the overall success rate of covalent, target-bound drugs has not been evaluated. While there have been numerous drugs withdrawn from the market or withdrawn from clinical investigation,9 there is no systematic analysis of the mechanism leading to the toxicological event. There are several examples, however, that suggest that withdrawal is due to a promiscuous reactive metabolite, such as amineptine and sulfamethoxazole.10 The purpose of this study was to examine known covalent targetmodifying small molecule drugs and attempt to glean the characteristics of successful strategies from them. This review consists of a survey of covalently modified drug targets and reviews the types of chemical reactions employed therein. A critical eye has been given to identifying where covalent inhibition may be appropriately applied to early stage drug discovery programs.

Discussion Small molecule drugs and late stage clinical candidates that modulate their targets through covalent interactions can be divided into different categories as depicted in Table 2. These molecules react via one of the following reaction types: acylation, alkylation, metal/metalloid binding, disulfide/seleno bond formation, hemiketal formation, Michael addition, and the Pinner reaction. In most instances the small molecule interacts directly with its target while a subset of molecules undergo a transformation before reacting. These molecules display both reversible and irreversible binding characteristics, as well as slow release from the enzyme. The structures of the small molecules (Figure 2) are as diverse as the numerous clinical indications that these therapeutics target. I. Acylation Mechanisms. Triacylglycerol Lipase. Orlistat (1a) affects obesity management by inhibiting the body’s ability to metabolize dietary fats. Triacylglycerol lipase, a serine hydrolase, is secreted from the pancreas. Orlistat inhibits triacyglycerol lipase covalently by reversibly binding to the active site serine, with assistance from the histidine and aspartate residues comprising the catalytic triad (Scheme 1).11 The detailed mechanism has been elucidated from in vitro degradation studies of 14C-labeled orlistat bound to porcine pancreatic lipase. Animal studies indicate that orlistat is minimally absorbed from the gastrointestinal tract, with negligible concentrations in the plasma. Since orlistat is a potent inhibitor of a large number lipases, localization of the drug’s action to the lumen while minimizing systemic exposure is key to the overall safe profile of the drug.12 Serine-Type D-Ala-D-ala Carboxypeptidase. Drugs targeting serine-type D-ala-D-ala carboxypeptidase are used as broadspectrum antibacterial agents. Serine-type D-ala-D-ala carboxypeptidase is a serine protease involved in bacterial synthesis of the peptidoglycan layer in the cell wall. β-Lactam antibiotics are thus effective at weakening the cell wall of the bacterium, resulting in osmotic lysis and cell death. All β-lactam antibiotics irreversibly acylate the active site serine forming a serine-ester linked adduct 3 (Scheme 2).13 There are many β-lactam-derived antibiotics on the market targeting serine-type D-ala-D-ala carboxypeptidase, including amoxicillin (1b), ampicillin, and penicillin (Figure 3). Although this drug class is generally well tolerated, the rate of allergic reaction can occur in as high as

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1233

Table 1. Toxicological Effects That Can Result from Covalent Binding response anaphylaxis blood dyscrasis hepatic reactions cutaneous reactions

comments

immune mediated

direct effect on cellular function

Immediate response upon secondary exposure to antigen. Cause: rapid histamine release with accompanying vasodilation, blood pressure drop. Relatively uncommon. Manifests as aplastic anemia, agranulocytosis, hemolytic anemia. Range of responses from elevated serum transaminase levels (e.g., ALT, AST) to liver failure. Rarely associated with hypersensitivity. Responses range from mild (rash) to severe (Stevens-Johnson syndrome, toxic necrolysis). Included are delayed-type hypersensitivity reactions (DHR) and phototoxicity.

yes

no

yes

no

yes

yes

yes

no

Table 2. Targets, Indications, and Mechanism of Action of Covalently Interacting Small Molecules mechanism acylation

alkylation

metal/metalloid binding disulfide bond formation (seleno-enzyme) hemiketal formation Michael addition

target serine-type D-Ala-DAla carboxypeptidase triacylglycerol lipase acetylcholinesterase β-lactamase prostaglandin endoperoxidase synthase vitamin K epoxide reductase (warfarin-sensitive) enol-acyl carrier protein reductase aldehyde dehydrogenase UDP-Nacetylglucosamine-1carboxyvinyltransferase alanine racemase GABA-ATf aromatase proteasome H+/K+ ATPase P2Y12 purinoceptor antagonist thyroxine 5′deiodinase (type 1) serine protease hepatitis C virus NS3g ribonucleoside diphosphate reductase thymidylate synthase ErbB1/2g 5-R-reductase MAO-B

Pinner reaction

g

DPP IV cathepsin Kg

indication

name of drug or representative druga

reacting functionality

dose (mg)b

reversibility

bacterial infection

amoxicillinc

β-lactam

irreversible

100-500

obesity Alzheimer’s bacterial infection pain

orlistat rivastigmine clavulanatec aspirin

lactone carbamate β-lactam ester

reversible reversible irreversible reversible

360 6-12 500 1000

anticoagulant

warfarin

coumarin

bacterial infection (tuberculosis) alcoholism

isoniazid

hydrazided

irreversible

300

disulfiram

disulfide

irreversible

500e

bacterial infection

fosfomycin

epoxide

3000

bacterial infection (tuberculosis) epilepsy breast cancer multiple myeloma gastresophageal reflux disease platelet aggregation inhibitor hyperthyroidism

D-cycloserine

amined

>250

vigabatrin exemstanec bortezomib omeprazolec

amined methyl boronic acid sulfenamide

irreversible irreversible reversible irreversible

3000e 25 3 20

clopidogrel

thiol

irreversible

75

propylthiouracil

thiourea

viral infection

VX-950 (1q)

ketoamide

cancer

gemcitabinec

vinyl ketone

cancer cancer (NSCLC) benign prostatic hyperplasia Parkinson’s diseasei

floxuridinec HKI-272 (1t) finasteridec

diabetes osteoporosis

vildagliptin odanacatib

unsaturated amide unsaturated amide unsaturated amided aceylenic imined nitrile nitrile

selegilinec

2-10

450 reversible

n/a g150-000h

reversible irreversible reversible

0.1-0.6 (mg/kg)/d n/a 5

irreversible

1

reversible reversible

100 10-50j

a Prodrugs are indicated in italics. b As determined from the FDA label or other medical references. c Because of the large number of drugs developed for these targets, one representative drug is indicated in the table. d Indicates functionality covalently modified by the cofactor. e Estimated dose. f Approved in Canada, U.K., and Mexico. g Under clinical investigation. h Dose ) 1000 mg/m2 weekly. The average body surface area of a person is approximately 1.5-2 mm2. i Several irreversible MAO inhibitors are on the market for the treatment of depression. j Weekly dose used in the clinical trial “MK0822 (Odanacatib) Late Phase II Dose-Finding Study” described at www.clinicaltrials.gov.

18% of patients.14 Interestingly, it has been shown that factors in the serum facilitate the covalent binding of penicillin to serum proteins rather than inherent reactivity of the β-lactam with the proteins directly.15 Most certainly, the short treatment period aids in the tolerability of these agents. β-Lactamase. β-Lactamase inhibitors are used in conjunction with serine-type D-ala-D-ala carboxypeptidase inhibitors to treat

bacterial infection. Bacteria produce the serine hydrolase β-lactamase to hydrolyze carboxypeptidase inhibitors, forming a hydrolytically labile, fast turnover serine-ester linked adduct.16 However, reactions between β-lactamase and specific β-lactamase inhibitors such as clavulanate (1c) form kinetically stable irreversible adducts (4), functionally inactivating the bacterial resistance mechanism (Scheme 3). There are several β-lactamase


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Figure 2. Chemical structures of the compounds described in Table 2. Structures in green indicate prodrugs.

inhibitors on the market with the similar chemotypes including clavulanate (1c), tazobactam, and sulbactam (Figure 4). Acetylcholinesterase. Acetylcholinesterase (AChE) inhibitors are used to treat neurological diseases, including Alzheimer’s disease, by preventing the breakdown of acetylcholine and therefore preserving synaptically relevant concentrations of this key neurotransmitter. Rivastigmine (1d) is a slow-binding, reversible17 inhibitor of the serine hydrolase, AChE. Crystallographic data of rivastigmine bound to Torpedo californica AChE show the formation of the active site serine-carbamate adduct, assisted by other members of the catalytic triad. The leaving group phenol is retained in the “anionic site” of the enzyme in proximity to the site of adduction.18

Apart from the covalently bound rivastigmine (1d), donepezil and galantamine are competitive inhibitors of AChE also marketed to treat Alzheimer’s disease (Figure 5). On the basis of analysis of the side effects observe for each of these drugs, there does not seem to be a significant safety drawback to using a covalent versus noncovalent AChE inhibitor. Prostaglandin Endoperoxidase Synthase. Aspirin (acetylsalicyclic acid, 1e) is a widely used prostaglandin endoperoxidase synthase (cyclooxygenase) inhibitor. The primary indication is pain management, but at low doses aspirin is also widely used for cardiovascular benefit.19 Incubation and further degradation studies of [3H]aspirin with prostaglandin endoperoxidase synthase indicate that aspirin irreversibly

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Scheme 1. Depiction of the Reversible Inhibition of Triacylglycerol Lipase by Orlistata

a

The catalytic triad (Asp176, His263, and Ser152) is also illustrated.

Scheme 2. Depiction of the Acylation of Serine-Type D-Ala-D-ala Carboxypeptidase by Amoxicillin

acylates an active site serine.20 Adverse events associated with aspirin use include an increased risk of gastrointestinal bleeding21 and the development of Reye’s syndrome in children. Although there are many known noncovalently bound cyclooxygenase inhibitors, aspirin is the only covalent modifier on the market. Vitamin-K Epoxide Reductase (Warfarin-Sensitive). Thromboembolic disease is a condition derived from formation of blood clots that obstruct blood flow. Warfarin (1f) is an oral anticoagulant used for the treatment of venous and arterial thromboembolic disorders. Since Warfarin-treated microsomes do not recover vitamin K epoxide reductase activity after being washed, it has been concluded that the binding is irreversible.22 Although the mechanism of covalent binding is not well elucidated, a proposed mechanism involves opening of the coumarin ring by either a cysteine or neighboring (unidentified) nucleophilic residue (Scheme 4).23 Enol-Acyl Carrier Protein Reductase. Enol-acyl carrier protein reductase is an NADH-dependent enzyme essential for mycolic acid biosynthesis, an important component for the synthesis of mycobacterial cell walls. The enol-acyl carrier protein reductase inhibitor isoniazid (1g) is a first-line antituberculous agent used in the treatment of tuberculosis. In the

Figure 3. Examples of some β-lactam inhibitors of serine-type D-alaD-ala carboxypeptidase.

Scheme 3. Depiction of the Acylation of β-Lactamase by Clavulanate

proposed mechanism, catalase peroxidase converts isoniazid to a reactive isonicotinic acyl anion (5), which attacks NAD (6) to generate the isonicotinic acyl-NADH adduct (7) (Scheme 5).24 Whether the mechanism proceeds through a cationic or radical pathway, isoniazid forms a reversible enzyme-bound cofactor adduct (7), as determined by X-ray crystallography and mass spectrometry. Isoniazid is a known CYP inhibitor25 and is associated with liver injury ranging from minor nonspecific changes in hepatic structure to fulminant hepatic failure.26 The liver damage is proposed to occur through a metabolite, acylhydrazine, which is a different chemical species from the acyl intermediate 5 generated for covalent adduction. Although liver damage can be a serious effect of treatment, there is a medical need warranting the continued use of this drug. Aldehyde Dehydrogenase. The aldehyde dehydrogenase inhibitor disulfiram (1h) is used to treat alcohol abuse by causing a buildup of acetaldehyde, a metabolite of ethanol.27 Elevated levels of acetaldehyde result in a phenotype similar to that of an allergic reaction, with nasal congestion and flushing, therefore deterring alcohol consumption. Disulfiram is a time-dependent irreversible inhibitor of aldehyde dehydrogenase. Disulfiram, which acts as a prodrug, is believed to inhibit the enzyme by the mechanism shown in Scheme 6. The disulfide bond is cleaved and methylated to give the methyl dithioate (8). Compound 8 is oxidized to the methylsulfinyl methanethioamide (9), which forms an adduct with Cys302 (10). Two adducts have been isolated, indicating covalent modification by both the dialkyl (10) and monoalkylated intermediates (10′).28 II. Alkylation Mechanisms. Several alkylation mechanisms have been used to achieve covalent modulation; however, preactivation of the substrate prior to the alkylation event is required. UDP-N-Acetylglucosamine Enol-Pyruvyl Transferase. UDP-N-acetylglucosamine enol-pyruvyl transferase (or MurA) is an integral protein to the synthesis of peptidoglycans required for bacterial cell wall synthesis. Naturally occurring fosfomycin (1i) is a broad-spectrum antibiotic used to treat urinary tract infections. As determined by X-ray crystallography, fosfomycin irreversibly inhibits MurA by alkylating the catalytically


Scheme 4. Proposed Mechanism for the Inhibition of Vitamin K Epoxide Reductase by Warfarin

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Scheme 5. Proposed Mechanism for Formation of the Isoniazid-NADP Adduct

Figure 4. Examples of some β-lactam inhibitors of β-lactamase.

Figure 5. Covalent and competitive AChE inhibitors used to treat Alzheimer’s disease.

important Cys115.29 Crystallographic studies have also shown a large protein conformational change upon binding fosfomycin. It is proposed that this conformational change facilitates nucleophilic attack on the epoxide by Cys115 to generate the enzyme-bound inhibitor. Alanine Racemase. Alanine racemase is a pyridoxal 5′phosphate (PLP) assisted enzyme that catalyzes the conversion of L-alanine to D-alanine, a constituent required for the synthesis of the peptidoglycan layer of the bacterial cell wall. DCycloserine (1j) is a broad-spectrum antibiotic used as a secondline treatment of tuberculosis. D-Cycloserine irreversibly inhibits alanine racemase by covalently modifying the PLP cofactor, with subsequent noncovalent enzymatic binding of the PLPcycloserine adduct 14 (Scheme 7).30 The irreversible inhibition was determined from enzyme-substrate time-dependent inactivation studies. The inhibition mode was determined by a crystal structure of adduct 14 bound to the enzyme. A proposed mechanism for formation of adduct 14 involves the initial formation of an iminium species between the aldehyde of PLP and Lys 39 (11). Subsequent transamination with D-cycloserine 1j followed by tautomerization and deprotonation forms a stable aromatic isoxazole moiety (14 in Scheme 7). Although Dcycloserine acts as an irreversible inhibitor of many PLPdependent enzymes and is thus a less selective drug, there is a medical need warranting continued use. γ-Aminobutyric Acid Aminotransferase. γ-Aminobutyric acid aminotransferase (or GABA-AT) is a PLP-dependent enzyme that catalyzes the catabolism of the inhibitory neu-

rotransmitter GABA. Vigabatrin 1k irreversibly inhibits GABAAT and is used (with restriction) to treat epilepsy in adults and children in Canada, the U.K., and Mexico, as it is associated with visual field defects in ∼40% of patients with long-term use.31 Likely because of the long-term effects on the retina, the FDA has not approved its use for epilepsy. However, vigabatrin is currently under phase II investigation for treatment of cocaine addiction at lower doses.32 Although vigabatrin is sold as a racemate, the (S)-enantiomer is the eutomer.33 Incubation of GABA-AT (isolated from a pig liver) with a radiolabeled vigabatrin confirms the irreverisbile binding and identified the site of modification at the lysine that normally binds the cofactor.34 After denaturation of the inactivated enzyme, the PLP cofactor was modified to pyridoxamine phosphate. Although the proposed mechanism of irreversible inhibition involves the PLP cofactor (Scheme 8), it is proposed to occur by a mechanism different from that of D-cycloserine with PLP35 (Scheme 7). It is postulated that vigabatrin forms an iminium species with PLP and tautomerizes to an activated enamine type species 15. Lysine addition to the β-carbon and hydrolysis of the resulting iminium afford the covalent adduct 16 with simultaneous formation of pyridoxamine phosphate. III. Metal/Metalloid Binding. Aromatase. Estrogen plays an important role in breast cancer development. Aromatase, a member of the iron-containing CYP superfamily, is an enzyme that aromatizes androgens to estrogens. The lowering of circulating estrogen levels by use of aromatase inhibitors, such as exemstane (1l), is one treatment strategy employed for breast cancer therapy. The steroidal inhibitor 1l is a time-dependent inhibitor of aromatase.36 The mechanism for irreversible inhibition of exemstane is not known, but the mechanism with the preferred endogenous ligand, androstenedione, has been studied (Scheme 9).37 Androstenedione is oxidized to hydroperoxy alcohol, which binds to iron, forming a peroxy-enzyme intermediate 17. Adduct 17 is then oxidized and aromatized to

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Scheme 6. Inhibition of Aldehyde Dehydrogenase by Disulfiram

Scheme 7. Mechanism for Formation of the PLP-Cycloserine Adduct 14

Scheme 8. Proposed Mechanism of Adduct Formation of Vigabatrin with PLP

Scheme 9. Inhibition of Aromatase by the Endogenous Ligand Androstenedione

the phenol 18, generating estrone. Although the final oxidation step’s details remain to be completely elucidated, it is known that aromatization proceeds with loss of the C19 carbon atom

as formic acid. On the basis of the structural similarities between exemstane and the endogenous ligand androstenedione, a similar mechanism can be proposed where upon formation of the


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Scheme 10. Inhibition of H+/K+ ATPase by Omeprazole

exemstane peroxy-iron intermediate (analogous to 17) aromatization does not occur, leading to irreversible inhibition (not depicted). Proteasomes. Cells regulate protein concentration and control the degradation of misfolded proteins by large protein complexes called proteasomes. Proteasomal inhibition by bortezomib (1m) is a mechanism for the treatment of blood cancers, multiple

Figure 6. Representation of the binding of bortezomib (shown in blue) in the active site of yeast 20S proteaseome. Hydrogen bonds are indicated by the dashed lines.

Figure 7. H+/K+ ATPase inhibitors omeprazole 1n, esomeprazole, and lansoprazole.

myeloma, and mantle cell lymphoma. Under physiological conditions, bortezomib primarily targets the proteasomal β5 active site of the 20S proteasome. The crystal structure of bortezomib with yeast 20S proteasome indicates a tetrahedral adduct between bortezomib and the active site threonine, while the free amino group of the threonine hydrogen-bonds to an acidic boronate hydroxyl group (Figure 6).38 The tetrahedral boronate adduct is further stabilized by a hydrogen bonding interaction between the second acidic hydroxyl boronate moiety with a neighboring glycine (stabilizing the oxyanion hole). This formation of a tetrahedral adduct is analogous to the action of the R-ketoamide VX-950 (1q) protease inhibitor. IV. Disulfide Bond Formation. H+/K+ ATPase. Proton transport by the gastric H+/K+ ATPase is the final step in gastric acid secretion. Proton pump inhibitors (PPIs) such as omeprazole 1n, esomeprazole, or lansoprazole (Figure 7) are used to treat gastresophageal reflux disease. Experiments with several radiolabeled PPIs indicate covalent inhibition of gastric ATPase via disulfide formation with one or more cysteine residues.39 The mechanism of PPI inhibition has been extensively studied.40 As illustrated with omeprazole, this class of inhibitors are prodrugs that transform under the acidic conditions on the stomach to a spiro intermediate 19 that undergoes aromatization to a sulfenic acid 20 followed by dehydration to a tetracyclic sulfeneamide 21. Compound 21 reacts irreversibly with a cysteine to form the disulfide adduct 22 (Scheme 10). The unstable reactive intermediate (20 or 21) is generated at the site of action, so the compound is efficacious and systemic exposure is not achieved. P2Y12 Purinoceptor. Platelet aggregation in occluded arteries can lead to stroke and myocardial infactions. Clopidogrel (1o) is a prodrug whose active metabolite inhibits platelet aggregation by irreversibly inhibiting the P2Y12 purinoceptor. The irreversibility of the active metabolite 24 has been determined in platelet binding studies.41 In the proposed mechanism, clopidogrel is oxidized by cytochrome P450 1A to a 2-oxo-thiophene 23 with

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Scheme 11. Proposed Mechanism of Clopidogrel Inhibition of P2Y12 Purinoceptor

Scheme 12. Proposed Mechanism for the Inhibition of Ribonucleoside Diphosphate Reductase by Gemcitabine

hydrolysis to a ring-opened carboxylic acid bearing thiol 24 (Scheme 11). It is proposed that 24 forms covalent adduct 25 via a disulfide bridge with a cysteine.42 Thyroxine 5′-Deiodinase (Type 1). Hyperthyroidism is caused by excess thyroid hormone production resulting in an overactive metabolism. Type 1 thyroxine 5′-deiodinase (or IDI) is a seleno-enzyme involved in the regulation of thyroid activity. Propylthiouracil (or PTU, 1p) irreversibly inhibits thyroxine 5′-deiodinase43 by formation of a covalent enzymeSe-PTU adduct (Figure 8).44 V. Hemiacetal Formation. Serine Protease Hepatitis C Virus NS3. Viral hepatitis causes acute inflammation to the liver tissue, which if left untreated can lead to a chronic condition that may progress toward liver cirrhosis or hepatocellular carcinoma.45 The hepatitis C virus NS3 serine protease ((HCV) NS3 · 4A) is proposed to be responsible for the virus’ RNA replication.46 VX-950 (1q) is currently under phase II investiga-

Figure 8. Thyroxine 5′-deiodinase-PTU adduct.

tion as a reversible (HCV) NS3 · 4A inhibitor. On the basis of modeling studies, 1q is proposed to inhibit serine protease hepatitis C virus NS3 by formation of a reversible covalent bond between the active site Ser139 and the ketoamide warhead (Figure 9).47 VI. Michael Addition. Ribonucleoside Diphosphate Reductase. Ribonucleoside diphosphate reductase is an enzyme catalyzing the formation of 2′-deoxyribonucletides ultimately used in DNA synthesis. Inhibitors of ribonucleoside diphosphate reductase, such as gemicitabine (1r), cladribine, or fludarabine

Figure 11. Thymidylate synthase inhibitors floxuridine 1s, capecitabine, and fluorouracil.

Scheme 13. Proposed Inhibition of Thymidylate Synthase by Floxuridinea

Figure 9. Proposed 1q-protein hemiketal adduct.

Figure 10. Ribonucleoside diphosphate reductase inhibitors gemcitabine (1r), cladribine, and fludarabine.

a

CH2FAH4 denotes the cofactor 5,10-CH2FAH4.


Figure 12. Schematic representation of the key interactions in the proposed binding model of 1t (shown in blue) bound to EGFR. Bonding distances (Å) are indicated by the dotted lines.

(Figure 10) are used to treat pancreatic and non-small-cell lung cancers. Studies have shown that the diphosphate of gemicitabine irreversibly inactivates the target in a time- and concentration-dependent manner in vitro.48 Although the site of covalent modification on the protein is unknown, gemicitabine reacts with a cysteine to generate the covalent adduct 28 via the vinyl ketone 27 (Scheme 12).49 Thymidylate Synthase. Thymidylate synthase generates thymidine monophosphate, which is subsequently phosphorylated and used in DNA synthesis and repair. Several uracilderived inhibitors of thymidlate synthase including floxuridine (1s), capecitabine, and fluorouracil (Figure 11) are used in the treatment of cancers. There is biochemical and spectral evidence that floxuridine binds covalently to thymidylate synthase in the presence of the cofactor CH2FAH4.50 Although it is known that the cofactor is converted to a different chemical species, the exact structure has not been elucidated. A proposed mechanism to account for the cofactor modification and the covalent enzymatic binding is depicted in Scheme 13. The reversible addition of a cysteine to the 6-position of the fluorouracil core (generating 29) is followed by reaction of the enolate with the cofactor (generating 30, Scheme 13). Epidermal Growth Factor Receptors. Epidermal growth factor receptor (EGFR or erbB1) and human epidermal growth factor receptor 2 (HER-2 or erbB2) are receptor tyrosine kinases whose overexpression is associated with a number of cancers. HKI-272 (1t) is currently under phase II clinical investigation as an irreversible dual erbB1/2 inhibitor for the treatment of non-small-cell lung cancers.51 The irreversibility of this chemotype has been demonstrated with an analogous compound, 2-EN-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide (EKB-569, an earlier clinical candidate) by incorporation of the compound into the EGFR protein upon incubation with A431 cells.52 Interestingly, EKB-569 was also shown to undergo Michael addition reaction of glutathione (19% conversion).53 The proposed binding model of 1t in HER-2 indicates that the Cys805 is 3.43 Å away from Michael acceptor β-carbon and is easily accessible for covalent interaction (Figure 12). The sulfhydryl hydrogen and the nitrogen of the dimethylamino group are situated for intramolecular catalysis.54 It is postulated that the Michael addition is accelerated because of the entropic effect of having the reactive center, nucleophile, and base catalyst in proximity. There are currently two noncovalent EGFR inhibitors on the market, gefitinib and erlotinibm, and one noncovalent erbB2 inhibitor, lapatinib (Figure 13). In addition to maintaining activity against mutations,55 covalent modification of the target is proposed to be beneficial over competitive inhibition because

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irreversible inhibition can occur in the presence of millimolar adenosine triphosphate levels within the cell and inhibition can only be overcome by new synthesis of the target, resulting in a sustained effect.56 5-r-Reductase. 5-R-Reductase is an NADPH-dependent enzyme that catalyzes the reduction of testosterone to dihydrotestosterone, the more potent androgen implicated in the development of enlarged prostates. Inhibitors of 5-R-reductase, such as finasteride (1u) and dutasteride (Figure 14), are used for the treatment of benign prostatic hyperplasia. 1u covalently inhibits 5-R-reductase by forming a reversible, enzyme bound adduct with the NADP cofactor (32). Hydride addition to the β-carbon of the vinylogous amide affords the dihydrofinasteride enolate 31, which attacks NADP+, forming the NADP-dihydrofinasteride adduct 32 (Scheme 14).57 Monoamine Oxidase B. Monoamine oxidase B (MAO-B) is an enzyme that is located in the mitochondrial membrane and is responsible for catalyzing the oxidative deamination of biogenic amines. Selegiline (1v) and rasagiline (Figure 15) are used to treat Parkinson’s disease by irreversibly inhibiting MAO-B and therefore inhibiting MAO-B mediated degradation of dopamine postsynaptic release.58 A covalent adduct with selegiline has been isolated and demonstrated to proceed through the isolloxazone moiety of the FAD cofactor (covalently attached via the C8 to a cysteine, as determined by absorption spectral analysis) (36 in Scheme 15).59 The proposed mechanism for the formation of 36 involves an initial single electron transfer (SET) to the flavin to generate an aminium radical 33. Subsequent steps have been suggested to occur either by Michael addition to the vinylogous iminiumyl cation intermediate (35 as depicted in Scheme 15) or by a radical coupling mechanism.60 Both mechanisms result in a Michael-type adduct 36. VII. Pinner Reaction. Dipeptidyl Peptidase IV. Diabetes is a disease involving the improper synthesis or use of insulin. Dipeptidyl peptidase IV (DPP-IV) is a serine protease that degrades the incretin hormone glucagon-like peptide 1 (GLP1), a peptide required for the glucose-dependent regulation of insulin. Inhibition of DPP-IV by vildagliptin (1w, Figure 16), approved for use in Europe and under phase III investigation, or saxagliptin increases the level of active GLP1, resulting in improved glucose tolerance. On the basis of the crystal structures of chemically related pyrrolidine nitriles with DPP-IV, it is believed that vildagliptin forms a reversible covalent imidate ester adduct with the active site serine (Ser610) likely assisted by protonation by a neighboring residue (Figure 17).61 A noncovalent DPP IV inhibitor, sitagliptin, was recently approved for use in the U.S. Sitagliptin binds to the same region of the protein as the pyrollidine nitrile compounds, with the amide carbonyl of sitagliptin binding to Tyr547.62 Cathepsin K. Cathepsin K (Cat K) is a lysosomal cysteine protease fundamentally involved in osteoclast mediated degradation of the bone. Inhibition of Cat K is proposed to aid in the prevention and progression of osteoporosis. Odanacatib (1x), currently under phase III clinical investigation, is described as a reversible covalent Cat K inhibitor.63 A published crystal structure of a chemically similar dipeptidic nitrile indicates a covalent bond between the nitrile and the active site Cys25 (Figure 18).64 The resulting thioimidate NH hydrogen-bonds with a neighboring carbonyl group. Perspective The examples presented herein provide strong evidence that covalent modifiers can be safe and effective therapeutics.

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Figure 13. EGFR inhibitors gefitinib and erlotinib and ErbB2 inhibitor lapatinib.

Scheme 14. Proposed Mechanism of Inhibition of 5-R-Reductase by Finasteridea

a

PADRP ) phosphoadenosine diphosphoribose.

Figure 14. 5-R-Reductase inhibitors finasteride (1u) and dutasteride.

Figure 15. MAO-B inhibitors selegiline 1v and rasagiline used to treat Alzheimer’s disease.

While in many instances the mechanism of inhibition was determined after efficacy was realized, one could adopt a covalent modifier approach from the beginning of a program. One key success factor for this approach is the proper selection of the warhead moiety. Although there are examples of compounds containing very active functionality, such as apirin 1e (activated ester) and fosfomycin 1i (epoxide), a majority of the successful drugs contain functionality whose reactivity is attenuated to achieve targeted modulation. For example, the binding of rivastigmine 1d to acetylcholinesterase activates the carbamate toward cleavage by the active site serine of the catalytic triad. Another elegant example is finasteride 1u, which acts as a selective hydride acceptor from NADPH only when bound to 5R-reductase.65 In addition, the Cat K inhibitor odanacatib 1x highlights the reversible nucleophilic addition of an active site thiol to a nitrile. These examples illustrate how the location of the warhead within

a structural motif can deliver both the desired therapeutic effect and safety profile. Additionally, the prodrug approach is also valid but arguably more challenging. There are several drugs that utilize a masked warhead as the electrophilic component such as the H+/K+ ATPase inhibitors (exemplified by omeprazole 1n), where the reactive species is generated in the acidic environment of the stomach where the drug exercises its antisecretory effect. This target-localized formation of the reactive intermediate reduces systemic exposure and potential for off-target toxicities.66 The blockbuster drug clopidogrel 1o is converted to an active metabolite that is hypothesized to react preferentially with P2Y12 to prevent stoke. Whether these successful drugs were discovered serendipitously or by design, we can use the insight provided by the available mechanistic and/or structural information to enable future de novo design of selective covalent modifiers. Paramount for success is the availability of detailed structural information on protein-ligand interaction, such as that derived from of X-ray crystallography, to facilitate the refinement of compound design and warhead placement. This approach is elegantly illustrated by the EGFR inhibitor 1t, where an appropriately placed Michael acceptor reacts readily with a nucleophilic amino acid side chain when facilitated by assistance from an internal basic amine moiety. A systematic review of the known covalently modulated targets reveals several trends (Table 3, Charts 1 and 2). It is no surprise that the most prevalent covalently modified targets identified are enzymes (Chart 1). As a subset of the overall targets, the cysteine and serine residues are primarily modified, with few examples of other nucleophilic amino acid residues (Chart 2). Among the enzymes, proteases or hydrolases appear frequently. In addition, cofactor mediated enzymes are also represented. These data indicate that cofactor mediated enzymes or enzymes bearing an active site cysteine or serine represent attractive targets for covalent modification. The strategy to drug a target through employing covalent modifying approach could provide advantages under certain


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Scheme 15. One of the Proposed Mechanisms for Formation of the FAD-Selegiline Adduct (Bound through N5 of the Isolloxazone Moiety)a

a

MAO-F indicates the MAO-flavin moiety.

Figure 16. DPP-IV inhibitors vildagliptin (1w), saxagliptin, and sitagliptin.

Figure 17. Representation of the crystal structure of a pyrrolidine nitrile bound to DPP-IV. It is predicted that vildagliptin and other pyrollidine nitriles bind in a similar fashion.

scenarios. There is typically a cost to improving the potency of lead structures that bind through noncovalent interactions. This endeavor must balance increases in molecule weight, lipophilicity, and hydrogen bonding functionality that can be detrimental to other important properties such as pharmacokinetics and ancillary pharmacology. In contrast, when a significant amount of binding energy is derived from the drug-protein covalent bond, there should be a reduction in the number noncovalent interactions needed to achieve desired potency. In the case of irreversible binders, drug concentrations in systemic circulation need only be available for a long enough period to

Figure 18. Schematic representation of the binding mode of a related Cat K inhibitor, shown in blue (based on the crystal structure). Hydrogen bonds are indicated by dotted lines, and values are given in angstrom.

achieve target coverage, potentially deemphasizing the need for a high, prolonged systemic drug load and therefore potentially mitigating off-target activity.67 Also, the half-life of the compound need not be long in order to achieve once a day or twice a day dosing. Certainly, reversible noncovalent inhibitors that display slow off-rates would also provide a similar benefit. While there will always be a healthy debate about pursuing molecules that bind covalently, this risk may be minimized by pursuing covalent modifiers that would be administered acutely or to patients with a life threatening disease. Analysis of the pharmacodynamic needs of a particular therapy may lead one to consider irreversible covalent inhibition. For many diseases pharmacodynamic activity is correlated to the degree of target inhibition or occupancy. For therapies that require a high target occupancy for effective treatment, such as cancer or antibacterial therapeutics (where in the absence of high target coverage mutations may occur),68 irreversible covalent modulation could be the most effective means of treatment. Conversely, there are therapeutic axes that would not benefit from complete covalent inhibition, wherein the complete shutdown of a primary pathway would lead to on-target toxicities. In these instances, irreversible covalent inhibition may not be appropriate. For example, in the case of warfarin, it is known that using the drug for an extended period of time (or at a high dose) can cause fatal bleeding. For this reason, warfarin is recommended for short-term use; when warfarin is used for long-term thrombosis therapy, patients are closely monitored.

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Chart 1. Graphical Representation of the Diversity of Target Types

Chart 2. Breakdown of Protein Functionality Modified by Covalent Drug

That said, the industry is still searching for a safe and effective alternative to warfarin.

Whether medicinal chemists pursue covalent or noncovalent modifiers, compounds should be selective for the desired target. This selectivity encompasses related pharmacological targets, as well as other endogenous nucleophilic moieties such as proteins, peptides (such as glutathione), and DNA. In any drug discovery program ancillary pharmacology studies are conducted to assess the potential liability for observing off-target toxicities in addition to in vitro safety studies. While selectivity criteria are identical for programs striving to develop either a covalent or noncovalent modifier, one might consider conducting studies to determine promiscuous binding earlier in a program utilizing a potentially reactive functional group. It is interesting to consider how an organization might become better positioned to exploit covalent modification as a more general approach to drug discovery. For instance, one may consider building a focused screening set that would be populated with low molecular weight compounds that possess “low to moderately” reactive functionality. A lead identified from this collection could be optimized with information from crystallography and modeling studies. Medicinal chemists could further “fine-tune” reactivity, if needed, so covalent adduction

Table 3. Targets of Covalent Inhibition target type serine proteases

serine hydrolases miscellaneous serine-containing enzymes cysteine proteases cysteine-containing enzymes

cysteine-containing GPCRs cofactor mediated enzymes seleno-enzyme metal-containing enzyme (Fe) other

target

functionality covalently modified by the protein

serine-type-D-Ala-D-Ala carboxypeptidase triacylglycerol lipase DPP IV serine protease hepatitis C virus NS3 acetylcholinesterase β-lactamase prostaglandin endoperoxidase synthase cathepsin K aldehyde dehydrogenase H+/K+ ATPase UDP-N-acetylglucosamine-1-carboxyvinyltransferase vitamin K epoxide reductase (warfarin-sensitive) ribonucleoside diphosphate reductase EGFR thymidylate synthase P2Y12 purinoceptor antagonist 5-R-reductase enol-acyl carrier protein reductase MAO-B alanine racemase GABA-AT thyroxine 5′-deiodinase (type 1) aromatase proteasome

β-lactam lactone nitrile ketoamide carbamate β-lactam ester nitrile disulfide sulfenamide epoxide coumarin vinyl ketone unsaturated amide unsaturated amide thiol unsaturated amide hydrazide aceylenic imine amine amine thiourea methyl boronic acid


is confined to the target protein. Of course opinions regarding an acceptable level of reactivity for a lead structure will always be defined differently throughout the industry. In addition, identification of functional groups beyond those mentioned in this review that selectively form covalent adducts could further enable this strategy. There are a number of covalent modifiers in preclinical or early clinical investigation that will continue to offer insight to this drug discovery strategy, including ones that target the caspases,69 MMP13,70 thyroid hormone receptor,71 and FAAH.72 Certainly, the presence of small screening or fragment sets comprising compounds with low to moderately reactive functionality will be crucial for providing starting points. Alternatively, one could look to strategically position a warhead within a lead compound. Hopefully, the compilation of examples in this review will inspire drug discovery scientists to consider pursuing covalent modifiers in the future. Acknowledgment. We thank J. C. Harmange, Richard Lewis, Yohannes Teffera, and Alan Cheng for fruitful discussions. In addition, we acknowledge Alessandro Boezio’s contribution to the orlistat portion of this review.

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Biographies Michele H. Potashman earned a B.A. degree in Chemistry (with honors) from the University of Pennsylvania in 1997 and a Ph.D. in Organic Chemistry in 2002 from the Massachusetts Institute of Technology under the guidance of Professor Stephen L. Buchwald. In 2002, she began her professional career at Amgen in Cambridge, MA. She is currently a Senior Scientist in the medicinal chemistry group focused on the discovery of novel agents for the treatment of cancer. Mark E. Duggan has over 20 years of experience in the pharmaceutical and biotechnology industry. Prior to joining Link Medicine as Vice President of Chemistry, Mark was Head of the Cambridge Massachusetts Medicinal Chemistry Department of Amgen. Before that, he spent 18 years at Merck & Co. where he was engaged in drug discovery projects in the areas of cardiovascular disease, bone biology, endocrinology, oncology, and neuroscience. Mark is a co-inventor of the antithrombotic agent, Aggrastat. He has authored over 50 publications and is an inventor on 52 patents. Mark holds a Bachelors degree in Chemistry from The Pennsylvania State University and a Ph.D. in Organic Chemistry from University of Pennsylvania under the tutelage of Professor K. C. Nicolaou.

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