'Rational Drug Design' is a newly coined concept which involves more and more of scientific logic in discovering new drugs. The process starts with the identification of the disease state, the biochemical processes involved in it, identification of the biomolecules which are to be targeted, designing in silico 'Leads' which would bind to these targets, synthesis of those leads and their bioscreening and ultimately optimizing these lead molecules to afford new acceptable drugs to cure that disease. Rational drug designing has been greatly helped by the phenomenal advancements in biological sciences, spectral techniques, robotic assay methods and development of computational softwares.
Till recently, researchers have been targeting the receptors or DNA for the treatment of various diseases. Now, in the form of enzymes we have new targets to cure a disease. Protease is a class of enzymes which are responsible for the hydrolytic breakdown of various proteins and polypeptides. They perform different physiological functions in digestion, fertilization, growth, differentiation, signaling / migration of cells, wound healing, apoptosis and in immunological mechanisms. These controls are performed by proteases in regulating protein synthesis and turn over of the synthesis. Not only that they lay important physiological role in healthy state in eukaryotes and prokaryotes they also play crucial role in propagation of inflammatory, respiratory, cardiovascular, neurodegenerative (Alzheimer's) and immunological (autoimmune) diseases. They have also been implicated in certain types of cancers, pancreatic diseases (malaria, schistosomia, leishmania, fungal) and viral infections (HIV, hepatitis, herpes).
So, these proteases are very attractive targets for the development of new drugs for the treatment of variety of ailments. Normal substrates for these proteases are proteins and polypeptides. In order to disrupt the functioning of these proteases, changes are to be carried out in their substrates in such a way that efficient binding to the target protease is achieved without giving the normal products. But, proteins or polypeptides as inhibitors of the proteases cannot be compromised due to certain limitations. When given by oral route they have poor bioavailability and membrane permeability. Due to non-selective proteolytic degradation their life span in blood stream or in cell is limited and they are susceptible towards fast elimination from the body. They have adverse pharmacokinetic parameters due to high order of their molecular weight. Even if the above parameters could be controlled they lack selectivity for a particular protease as the body is in rich in different types of proteases (cysteine, serine, aspartate and metalloprotease).
In order to circumvent all these problems nonpeptides are advocated to be developed as protease inhibitors. These compounds would have low molecular weight and possess no / minimum peptide bond character. It is known that the majority of the proteases bind to the substrates / inhibitors in extended ß-strand (ß -plated) conformation, so these non-peptides could have an entropic advantage in binding to the proteases if they have some conformationally restricted fragments as part of their structures. The field of development of nonpeptide protease inhibitors as potential drugs is wide open. Three dimensional structures of a large number of proteases are known and available in public domain. By direct drug designing using computer-assisted molecular modeling, nonpeptide molecules could be designed, synthesized and evaluated. Homology modeling and structure-based designing could be used in those cases where complete structure and binding sites of the proteases are not known. In fact, certain of the protease inhibitors have already hit the market as successful drugs for certain diseases.
Drugs in clinical use
A number of HIV-1 protease inhibitors are currently in clinical use. Drugs like saquinavir, ritonavir, indinavir, neflinavir and amprenavir are important constituents of cocktail therapy for HIV treatment. These protease inhibitors have been developled by substrate structure-based drug design. Another successful story of development of protease inhibitors as drugs is that of ACE-inhibitors as antihypertensive agents. ACE is a zinc metallprotease responsible for the formation of vasoconstricting agent angiotensin-II in the body from the decapeptide angiotensin-I. Inhibition of ACE would lead to decreased levels of angiotensin-II causing decrease in blood pressure. Captopril can be considered to be the first protease inhibitor developed on the principles of rational drug design. Other successful drugs from this class of protease inhibitors which are clinically used are enalapril, lisonopril, benazepril, ramipril, quinapril and fosinopril
Thrombin inhibitors: Thrombin is a serine protease that lays a crucial role in hemostasis and induces platelet aggregation. It is the final enzyme in the blood coagulation cascade. A number of active site-directed competitive inhibitors of thrombin are in different stages of clinical trials. These include argatroban, napsagatran, inogatran, efegatran, melagatran and LY178550.
Factor Xa inhibitors: Factor Xa is a trypsin-like serine protease responsible for cleaving prothrombin to form thrombin. Non peptide inhibitors of factor Xa have been developed. Although, none of these inhibitors has been approved as yet for clinical use. Some of them like DX-90659, YM-60828 and ZK-807191 are at different stages of development.
Neutral Endopeptidase Inhibitors: Neutral endopeptidase (NEP), a zinc metalloprotease is the main protagonist in the breakdown of the atrial natriuretic peptide (AVP) which raises urinary excretion of water and sodium and lowers plasma rennin and aldosterone levels. Inhibitors of NEP may have appli-cations as analgesic and antihypertensive agents. A number of these agents are under development out of which candoxatril is in the late stage of Phase-III for the treatment of congestive heart failure.
Dual ACE/NEP Inhibitors: Inhibition of ACE or NEP can lead to a reduction in blood pressure by reducing levels of angiotensin-II or increasing levels of ANP have led to the development of broader spectrum inhibitors that potently inhibit both enzymes and are thus more effective for the treatment of hypertension.
Such dual inhibitors have been shown to possess synergistic effects. Fasidotril, samapatrilat and omapatrilat are such compounds under clinical trials.
Cathepsin K Inhibitors: Cathepsins are intracellular cysteine proteases usually found in lysosomes and are active at acidic or neutral pH. Cathepsin K is selectively expressed in osteoclasts and has been proposed to play an important role in osteoclast-mediated bone resorption. Cathepsin K inhibitors are promising therapeutics for the treatment of diseases characterized by excessive bone loss such as osteoporosis. APC-33228 a vinyl sulfone is a suicide substrate in its preclinical stage of development for the treatment of osteoporosis.
Cathepsin L Inhibitors: Cathepsin L, a lysosomal cysteine protease secreted by osteoclast participates in bone resorption by degrading extracellular matrix proteins such as collagen. It is also implicated in tumor metastasis. An aldehyde derivative (12) is reported to be a first example of orally active cathepsin L inhibitor for preventing bone loss in ovariectomized animals.
Cathepsin B Inhibitors
Cathepsin B a lysosomal cysteine protease plays various digestive and processing roles inside cells to maintain normal cellular metabolism. However, when overexpressed it has been associated with several pathophysiological conditions such as tumor metastasis, inflammation, bone resorption and myocardial infarction. E-64 isolated from Aspergillus japonicus is a broad spectrum inhibitor of cathepsin B, L and calpain.
Cathepsin D Inhibitors: Cathepsin , an intracellular aspartic protease found in lysosomes performs 'house keeping' functions including degradation of cellular or phagocytosed proteins for reprocessing. The enzyme may be involved in a variety of disease states including cancer and Alzheimer's disease (formation of ß-amyloid peptide).
Pepstatin A remains the most potent inhibitor known for human cathepsin D.
Elastase Inhibitors
Human neutrophil elastase released in response to inflammatory stimuli is responsible for degradation of connective tissue proteins. Elastasin implicated in Adult Respiratory Distress Syndrome (ARDS), rheumatoid arthritis, pulmonary emphysema, cystic fibrosis and chronic bronchitis. Normally, elastase activity is tightly regulated by endogenous inhibitors but an imbalance between proteases and antiproteases can lead to degradation of healthy tissue leading to disease development. A number of elastase inhibitors are at different stages of development for various diseases. The trifluromethyl ketone derivative ZD8321 has shown oral bioavailability and excellent pharmacokinetics.
Tryptase Inhibitors
This is the major secretory product of human mast cells. This enzyme is released in response to mast cell activation along with histamine, heparin and other proteases. It has been implicated as an inflammatory mediator in different inflammatory and allergic conditions such as conjunctivitis, rhinitis and especially asthma. Compound showed good selectivity for tryptase over elastase and trypsin and was found to reduce oedema by 69 % compared to the controls in animal models.
Caspase Inhibitors
Caspases (ICE-like proteases) are important therapeutic candidates for the treatment of inflammatory disorders and neurodegenerative disorders. A number of inhibitors having chemical war-heads are under development stages.
Calpain Inhibitors: Calpains comprise a family of six distinct cytosolic cysteine proteases. They are calcium-activated and ubiquitously distributed in various cells. Their precise functions are yet to be elucidated. They have been postulated to be involved in protein turnover, protein kinase C activation, cytoskeleton and cell membrane organization and to interact with various membrane receptors and calmodulin-binding proteins. They are believed to play key role in the pathology of such disorders as stroke, Alzheimer's disease, muscular dystrophy, cataract and arthritis. Most of the inhibitors under developmental stage are active-site-directed inhibitors. However, their selectivity poses a big problem in their development as drugs.
Tumor Necrosis Factor a converting Enzyme (TACE) Inhibitors: The zinc metalloenzyme TACE cleaves a membrane-bound protein (pro-TNF-a) releasing TNF- a which plays an important signaling role in inflammatory disorders such as rheumatoid arthritis, multiple sclerosis and Crohn's disease. Very few studies have been directed to develop TACE inhibitors. Compound (17) has been claimed to be an inhibitor of TNF- a formation and soluble CD23, a protein associated with autoimmune diseases and allergy.
Matrix Metalloprotease (MMP) Inhibitors: MMP's are a group of structurally related metalloproteases which degrade and remodel structural proteins in the extracellular matrix such as membrane collagens, aggrecan, fibronectin and laminin. They are implicated in tissue remodeling at various stages of human development and wound healing. Currently seventeen human MMP's are known and they are involved in growth and spread of malignant tumors, development of chronic diseases such as multiple sclerosis, arthritis, fibrosis and other inflammatory conditions. Quite a number of MMP inhibitors are undergoing evaluation in clinical trials for the treatment of cancer, arthritis and multiple sclerosis. Of these marimastat is in the most advanced state.
Plasmepsins Inhibitors: Plasmepsin I and II, found in the malarial parasite P. falciparum are believed to be essential for the degradation of its major food source human hemoglobin. Inhibition of these enzymes is considered to be a viable therapeutic strategy for the treatment of malaria. All compounds developed till date as plasmepsin inhibitors are also found to be potent inhibitors of human cathepsin-D. So a better selectivity needs to be attained for their inhibitors. Falcipain found in the parasite also take part in protein degradation at some stage. Combined inhibitors of plasmepsins and falcipains are considered to be more effective in plasmodium-mediated hemoglobin degradation in both culture and murine malaria model. ..
(The author is with Pharmacy Department Faculty of Tech. & Engg., Kalabhavan The M.S University of Baroda)