K Inhibitors

Cathepsin K (Cat K) is a member of the CA1 family of lysosomal cysteine proteases. This family is comprised of 11 human members (cathepsins B, C, F, H, K, L, O, S, V, W, Z) which share a common papain-like structural fold and a conserved active site Cys-Asn-His triad of residues [1-3]. These enzymes are synthesized as pre-pro-enzymes and are converted from the catalytically inactive zymogen into the active form in acidic lysosomal environment. In some cases, cathepsins are also secreted in the active form from cells. The sequence identity of 

Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada H9H 3L1 

Annual Reports in Medicinal Chemistry, Volume 42 ISSN 0065-7743, DOI 10.1016/S0065-7743(07)42008-5 

The development of Cat K inhibitors, with an emphasis on the fundamental biology, pharmacology and human clinical trials has been recently reviewed [1-3]. The medicinal chemistry of Cat K inhibitors has also been the subject of recent reviews [7,8]. This article reviews recent publications and meeting abstracts on the design of Cat K inhibitors, as well as developments in Cat K-related biology including animal models for the prediction of inhibitor efficacy, the potential for the separation of bone resorption and formation by Cat K inhibitors, inhibitor selectivity considerations and other potential indications for Cat K inhibitors. 

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When the target enzyme is difficult to obtain, related enzymes could be used to provide insights in the design of novel ligands. For example, papain was used to design a class of potent cathepsin K inhibitors [33] spanning both sides of the papain active site. However, fine-tuning these inhibitors to produce more potent ones required the use of the crystal structure of cathepsin K itself [34],... 

LaLonde JM, Zhao B, Smith WW, Janson CA, DesJarlais RL, Tomaszek TA, Carr TJ, Thompson SK, Oh HJ, Yamashita DS, Veber DF, Abdel-Meguid SS. Use of papain as a model for the structure-based design of cathepsin K inhibitors crystal structures of two papain-inhibitor complexes demonstrate binding to S -subsites. J Med Chem 1998 41 4567-4576. 

The synthesis of the /V-protected 7-methylazepine derivative 34 was achieved in 89% yield by a ring-closing metathesis reaction on 33 mediated by Grubbs I ruthenium catalyst. This azepine was an important precursor for the preparation, via epoxidation of the double bond, of a number of 7-methylazepanone derivatives for evaluation as cathepsin K inhibitors <06JMC1597>. 

The standard model for the preclinical development of anti-osteoporosis therapies is the ovariectomized (OVX) rat. However, Cat K inhibitors developed specifically against the human enzyme are generally significantly less potent ( 2-orders of magnitude) against the rat and mouse enzymes than against human Cat K [9]. This loss of potency towards the rodent enzymes, which is consistent with their low sequence homology, therefore restricts the use of... 

A more long-term rabbit model of estrogen deficiency has also been recently described in which adult OVX rabbits (7 months) were treated with the Cat K inhibitor L-006235 (12) at 0,2,10 mg/kg, or ALN (0.125 mg/kg, 3x/wk) for 27 weeks. OVX resulted in an 11.5% vertebral bone loss compared to sham-operated controls. Both the high dose of L-006235 and ALN completely prevented this bone loss, whereas the low dose of L-006235 produced a partial response [19]. 

Metastatic bone disease (MBD) is characterized by very high levels of bone turnover in regions proximal to the tumour [33]. Bone resorption inhibitors such as bisphosphonates represent the current standard of care for the treatment of bone metastases primarily due to breast or prostate cancer and multiple myeloma. It has been proposed that other strong anti-resorptives such as a Cat K inhibitor could be useful in the treatment of bone metastases. Evidence for this has been presented in the form of a preclinical MBD model in which human breast cancer cells are implanted into nude mice. Treatment with a Cat K inhibitor gave a significantly lower area of breast cancer-mediated osteolytic lesions in the tibia [34]. In a separate study, the efficacy of a Cat K inhibitor in the reduction in tumour-induced osteolysis was found to be enhanced in the presence of the bisphosphonate zolendronic acid [35,36]. When prostate cancer cells were injected into the tibia of SCID mice, treatment with a Cat K inhibitor both prevented and diminished the progression of cancer growth in bone [37]. 

Cat K inhibitor therapy may also result in protection against the development of atherosclerosis. Cat K-deficient mice show reduced atherosclerotic lesion number and size on an ApoE receptor-deficient background, compared to wild-type animals [38,39]. Cat K is also associated with increased adiposity in humans [27,28] and may also play a role as a kininase, suggesting a role in blood pressure regulation [40]. Cat K has also been postulated to play a role in the pathology of rheumatoid arthritis [41,42],... 

The a-ketoamide group is a well-known electrophile for the inhibition of both serine and cysteine proteases. Introducing a pyrazole as the N-substituent of the ketoamide provides potent inhibitors of Cat K [50]. SAR exploration of the P2-P3 residues in a series of Cat K inhibitors led to the identification of the pyrrolidine... 

Clinical data for two Cat K inhibitors, balicatib and MK-0822, has been reported. Additionally, relacatib has been reported to be in clinical development [44], but no data have been disclosed. 

The past 3 years have seen tremendous advances in both the design of Cat K inhibitors and in our understanding of the effect of Cat K inhibition on bone remodeling. The structural diversity of Cat K inhibitors has expanded considerably from simple peptidomimetics to non-peptidic derivatives and even non-covalent inhibitors. The potency, selectivity and pharmacokinetic properties of key compounds are very attractive and seem well-suited to further development. The disclosure of clinical validation of the effect of Cat K inhibition on bone mineral density, plus the provocative data suggesting a decoupling of bone resorption and bone formation provides a compelling framework for further development of Cat K inhibitors for the treatment of osteoporosis. 

Constitutive activation of the P13-K - Akt/PKB survival signaling pathway is a likely mechanism by which many cancers become refractory to cytotoxic therapy. In LNCaP prostate cancer cells, the PTEN is inactivated, leading to constitutive activation of Akt/PKB and resistance to apoptosis. However, apoptosis and inactivation of Akt/PKB can be induced in these cells by treatment with P13-K inhibitors. Surprisingly, androgen, epidermal growth factor, or semm can protect these cells from apoptosis, even in the presence of P13-K inhibitors and without activation of Akt/PKB, indicating the activity of a novel, Akt/PKB-independent survival pathway. This pathway blocks apoptosis at a level prior to caspase 3 activation and release of cytochrome c from mitochondria (Carson et al, 1999)... 

Programmed cell death plays an important role during lymphocyte development, by eliminating autoreactive cells, as well as in the effector phase of the immune response, when antigen induced cell death halt cell activation. Several groups have been studied the function of PTEN in the immune response. PTEN heterozygous (PTEN+/-) mutants develop a lethal polyclonal autoimmune disorder with features reminiscent of those observed in Fas-deficient mutants. Fas-mediated apoptosis was impaired in Pten+/-mice, and T lymphocytes from these mice show reduced activation-induced cell death and increased proliferation upon activation. PI3-K inhibitors restored Fas responsiveness in PTEN+/- cells. These results indicate that PTEN is an essential mediator of the Fas response and a repressor of autoimmunity, thus implicate the P13-Kinase/Akt pathway in Fas-mediated apoptosis (DiCristofano et al, 1999)... 

Case study structure-based design of cathepsin K inhibitors... 

Figure 17.3 Schematic representation of the design of the symmetric cathepsin K inhibitor diacylaminomethyl ketone (1,3-bis[[A/-[(phenylmethoxy)carbonyl]-L-leucyl]amino]-2-propanone), based on the crystal structures of papain bound to leupeptin (Leu-Leu-Arg-aldehyde) and to Cbz-Leu-Leu-Leu-aldehyde, and an example of its further optimization.

Table 17.2 Structure-based enhancement of cathepsin k inhibitor s potency and selectivity...

Thompson et ah, 1998 Marquis et al., 1999). Once each half was optimized, more SBDD cycles were needed to tweak the full molecule. This work resulted in numerous potent and selective cathepsin K inhibitors an example is shown in Fig. 17.3. Improvements in potency and selectivity of the compounds shown in Fig. 17.3 are listed in Table 17.2. Much of this work was recently summarized in Veber and Cummings (2004). 

D. F. (1998). Stmcture-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic. /. Med. Chem. 41,3923-3927. 

Veber, D. F. and Cummings, M. D. (2004). Structure-based design of cathepsin K inhibitors. In Protein Crystallography in Drug Discovery. Methods and Principles in Medicinal Chemistry, Babine, R. E. and Abdel-Meguid, S. S., eds, Vol. 20, Wiley-VCH, p. 127-146. 

S. Leger, F. Masse, M.E. McGrath, D.J. McKay, M.D. Percival, D. Riendeau, S.B. Rodan, M. Therien, V.L. Truong, G. Wesolowski, R. Zambonia, W.C. Black, Identification of a potent and selective non-basic cathepsin K inhibitor, Bioorg. Med. Chem. Lett. 16 (2006) 1985-1989. 

The efficiency of inactivation by covalent bond formation vs release of the reactive species into solution has been described by its partition ratio. The most efficient inactivators have catalytic partition ratios of 0, in which case each inhibitor molecule leads to inactivation of the enzyme. To this date, many of these inhibitors have been designed, and alternative names like suicide substrate, Trojan Horse inactivator, enzyme induced inactivator, k inhibitor, and latent inactivator have been used for this class of inhibitors. A number of comprehensive reviews are available (26—32). 

A concise alternative synthesis of the azepinones 241 has been developed based on the key ring-closing metathesis of the a-amino enones 240 (Equation 34) <2006H(67)549>. The substrate concentration was 5 x 10 3M and yields of products are given in Table 4. The azepinone 241c was then converted to a known cathepsin K inhibitor. 

Actually, the mechanism underlying the effects of BEO on Akt phosphorylation were recently investigated in an in vitro model of excitotoxic insult, that is exposure of human neuroblastoma cells to NMDA (Corasaniti et al, 2007). We observed that neuroprotection afforded by BEO against NMDA-induced cell death is antagonized in a concentration-dependent fashion by LY294002, a specific PI3-K inhibitor (Vlahos et al, 1994). In addition, LY294002 inhibited the ability of... 

Treatment with the PI3-K inhibitor wortmannin reduces the number of surviving RGCs after retinal ischemia/reperfusion suggesting that, Akt activation is indeed endowed with RGCs neuroprotective properties and represents a prosurvival response of the retina to the ischemic injury (Russo et al., 2008a). 

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