Introduction

Proline Peptidase Overview

History

Dipeptidyl Peptidase IV's role in the inactivation of incretin hormones was discovered in the 1990s. Animal studies were conducted in the late 1990s, followed by human studies in the early 2000s. The first DPP-IV inhibitors (sitagliptin, vildagliptin, alogliptin, saxagliptin, and linagliptin) were approved starting in 2006, and now serve as monotherapy or add-on to other therapies in a glucose-lowering capacity. ^[1] Since their approval, there have been multiple long-term trials to continue exploring the long-term effects of these medications. It is known that gliptins directly impact the pancreas, kidney, heart, and vessels. The most investigated thus far is the effects of gliptins on renal and cardiovascular functioning. ^[2] Results of phase II and III trails indicated that gliptins did not harm the cardiovascular system. A meta-analysis implicated two possible beneficial effects for patients treated with these medications: a reduction in cardiovascular effects and a direct renoprotective effect. ^[3]

Function

DPP-IV, a moonlighting protein, has been implicated in many functions and diseases of the body including glucose metabolism, cardiovascular disease, the stress response, autoimmune diseases (i.e. HIV/AIDS), inflammation, and tumor biology. ^{[4] [5] [6]} The active site functions in two ways: it can bind inhibitors and it can truncate substrates. Inhibitors inhibit DPP-IV via competitive inhibition, binding to the active site but are not degraded, so they remain bound and block the enzymatic activity. Substrates are truncated by DPP-IV by temporarily forming a covalent bond and ultimately being released in two pieces (the first two residues and the truncated protein).

Structure

Overview

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DPP-IV is found in two forms in the body: a membrane bound monomer and a . All structural renderings of DPP-IV start at the 39th residue, meaning it does not include the intracellular domain, transmembrane region, and part of the cleavage site. The monomer has 4 domains: DPP-IV cleavage stalk, beta propeller, cystine-rich region, and the catalytic domain.

The DPP-IV beta propeller is notable as it differs from all the other enzymes in the dipeptidyl peptidase family. In all other DPPs the beta propeller has ligand gating potential; however, the is an asymmetrical 7 blade propeller that does not function as a ligand gate by rather acts as a binding site which allows DPP-IV to conjugate with Adenosine Deaminase. ^[7]

The contains 6 cystine residues (C385, C394, C444, C447, C454, C472) that make that play a critical role in the tertiary structure and therefore function of the DPPIV enzyme. ^[8]

The catalytic domain is where DPP-IV substrates are cleaved at he penultimate point or where inhibitors bind to prevent DPP-IV enzymatic activity.The includes the S1 binding pocket and S2 binding pocket. The S1 binding pocket contains hydrophobic residues (W547, S630, Y631, V656, W659, Y662, Y666, N710, V711 and H740) that interact with either the substrate or inhibitor to keep it within the catalytic domain. The S2 binding pocket contains residues (E205, E206, Y662, S209, R358 and P357) that create hydrogen bonds with either substrate or inhibitor to additionally keep it in place to be cleaved by the catalytic triad. Within these is the , assisted by the oxyanion hole (Y631). ^[9]

^[10]

Mechanism

Once a substrate is bound in the active site, DPP-IV utilizes a covalent catalysis mechanism to cleave the substrate at the penultimate position. Asp708 of the catalytic triad (Ser630, His 740, Asp708) pulls electron density from His740, allowing the histidine to pull electron density from Ser630, making serine a stronger nucleophile. The catalytic triad is assisted in this process by the oxyanion hole (residue Y631), providing stability and keeping the substrate in place. The water molecule attacks the carbonyl carbon, breaking the newly formed covalent bond, and releasing the first two residues of the starting substrate. The active site resets.

Figure 5. Mechanism for the cleavage of substrate of DPP-IV via the catalytic triad.

Inhibitors

Gliptins, a class of oral antidiabetic medications, are DPP-IV inhibitors. The Food and Drug Administration (FDA) has approved the following: sitagliptin, alogliptin, saxagliptin, and linagliptin. The European Medicines Agency (EMA) has approved all of those aforementioned in addition to vildagliptin. Each gliptin is a small molecule (≤ 1000 Da).

Relevance

DPP-IV is known to cleave dozens of peptides including, but not limited to, regulatory peptides, neuropeptides, and chemokines. DPP-IV substrates are between 20 and 100 residues long, all of which contain a penultimate proline or alanine, indicating a stereochemical preference (though other penultimate residues are known to be cleaved but with reduced catalytic efficiency).

Diabetes

Diabetes mellitus is a metabolic disorder, characterized by hyperglycemia, caused by irregularity in insulin secretion, insulin action, or a combination of both. Type 1 diabetes mellitus (T1DM) is characterized by the autoimmune destruction of pancreatic beta-cells, leading to diabetic ketoacidosis. Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and subsequent deficiency. ^[11]

(GLP-1) is a 30 amino acid hormone secreted into the gut by intestinal epithelial L-cells. GLP-1 is secreted in response to meal intake and is responsible for stimulating insulin production. GLP-1 binding to the GLP-1R located on the membranes of pancreatic cells results in the upregulation of insulin production and secretion which directly results in the lowering of blood glucose levels. ^[12]

DPP-IV binds and degrades GLP-1 resulting in heightened blood glucose levels. Insufficient GLP-1 production or signaling in response to meal intake is clinically associated with T2DM and morbidity. Since GLP-1 is a potent regulator of blood glucose levels, and DPP-IV antagonistically regulates GLP-1, this makes DPP-IV inhibitors an excellent candidate for pharmacological therapeutics for T2DM. ^[13]

is a 30 amino acid hormone which is secreted into the gut by intestinal epithelial L-cells.[12] GLP-1 is secreted in response to meal intake and is responsible for stimulating insulin production. GLP-1 is a major peptide which regulates blood glucose levels and is also a major contributor to DPPIV substrates. The concentration of active GLP-1 is tightly regulated by DPPIV. GLP-1 is rapidly metabolized and degraded by DPPIV before even reaching the gut, which results in increased blood glucose levels. Insufficient GLP production or signaling in response to meal intake as been clinically associated with Type 2 diabetes and morbidity given the close antagonist correlation between GLP production and blood glucose levels. The antagonistic regulation of GLP-1 by DPP-IV has made DPP-IV inhibition a very excellent candidate for pharmacological therapeutics. [14]