The First Reciprocal Activities of Chiral Peptide Pharmaceuticals: Thymogen and Thymodepressin, as Examples

Significant advances in molecular biology and bio-organic chemistry have changed a new drug research paradigm to treat various pathologies. Cell biology methods allow the reprogramming of specialized differentiated cells to return them to their native state [ 1 , 2 ]. Several dipeptides have been separated from the thymus extract and tested for immunoregulating properties [ 3 ]. Section 4 and Section 5 and the Discussion discuss the influence of peptides’ and proteins’ chirality and reciprocity on the future progress of D-biology. The discovery of the biological activity of immunotropic exogenous enantiomeric peptides made it possible to study their action on hematopoiesis and immunogenesis through stimulation or suppression of the body’s response. Our studies have confirmed the different influences of the chemical and optical configuration of each dipeptide constituent amino acid on the direction and intensity of biological activity. The number of modified proteins, monoclonal antibodies, peptides, and peptidomimetics developed and approved as a medication for many diseases is growing [ 4 ]. However, peptides are biochemically and therapeutically distinct from these groups [ 5 , 6 ]. The FDA defines peptides as polymers composed of 40 or fewer amino acids. They lie between small molecules and biotherapeutics and can combine both areas [ 7 , 8 ]. Since the discovery of chirality in living organisms, scientists have been trying to create enantiomeric D-peptides, D-proteins, and L-RNA polymerases, which produce D-proteins. To introduce the D-amino acid into the peptide sequence in living organisms, the enzymatically post-translational conversion of L-amino acid into a D-isomer transforming peptide sequence is carried out. Numerous applications for D-polypeptides and D-proteins could be expected. Still, so far, this is challenged by the difficulty in their preparation, particularly when their complexity increases with their size, folding, and post-translational modifications [ 9 ]. The modern mirror-image phage display (MIPD) approach has been developed to create a sequence entirely composed of D-amino acids [ 10 ]. To create functional mirror-image biology systems, an imperative step is to establish a chiral inverted version of the central dogma of molecular biology [ 11 ] or use non-ribosomal peptide synthetases (NPRSs) [ 12 ]. The first milestone will be the complete assembly of an enantiomeric ribosome comprising D-proteins and L-rRNAs. The ability to translate L-mRNA into D-proteins will be genuinely revolutionary [ 13 , 14 ]. D-peptides can show significant selectivity and potency and could be an excellent candidate for treating various diseases and human disorders. A wide range of experiments showed high immunostimulating activity for the dipeptide Glu-Trp. At the same time, the synthetic enantiomeric peptides D-Glu-D-Trp and D-Glu(D-Trp) expressed immunosuppressive activity [ 3 ]. Many proteinases in the body complicate the isolation of low-molecular-weight peptides from biological raw materials. It is tough to isolate dipeptides since proteinases contain dipeptidases that selectively hydrolyze dipeptides to amino acids [ 15 ]. This review provides data on the experimental development and clinical use of two enantiomeric drugs and Thymodepressin, created based on the dipeptides L-Glu-L-Trp (Thymogen) and D-Glu(D-Trp) (Thymodepressin), respectively, exhibiting immunostimulating and immunosuppressive properties.

This section provides data on the activity of immunosuppressants, regardless of their chemical structure. Immunosuppressants suppress the pathological immune response, mainly through a cytostatic effect on lymphocytes in the early stages of the immune reaction, followed by the action of cytokines produced during hyperimmunization. The immune system perceives the transplanted organ as a foreign object, and almost everyone who receives an organ transplant must take immunosuppressive drugs. Immunosuppressants help the transplanted organ remain healthy and undamaged [ 24 , 25 ]. 2.2.1. Calcineurin Inhibitors Cyclosporine is the first peptide immunosuppressor exhibiting a cytostatic effect on T lymphocytes that inhibits calcineurin, the interleukin-2 gene transcription. The discovery and use of Cyclosporine opened a new era in solid organ transplantation. Cyclosporine binds cyclophilins inside cells and forms a drug–receptor complex, inhibiting the nuclear factor of activated T cells (NF-AT) [ 26 ]. Acute and chronic nephrotoxicity is the main side effect of Cyclosporine and can be managed to a drug-regulated target level. Other side effects include hirsutism, gingival hyperplasia, neurotoxicity such as seizures and tremors, hypertension, and diabetes [ 27 ]. Tacrolimus is another calcineurin inhibitor that became available for clinical use in 1994 for renal and liver transplantation [ 28 ]. This drug binds to intracellular FKBP12, forming a drug–receptor complex that competitively binds with calcineurin and acts through the same pathway as Cyclosporine to inhibit T-cell activation and proliferation. Tacrolimus shows a similar immunosuppressive level with less toxic side effects [ 29 ]. The side effect profile for tacrolimus is identical to cyclosporine’s, with fewer hypertension complications [ 30 ].

Structurally similar to calcineurin inhibitors, mTOR inhibitors act by forming a drug–protein complex. However, they bind to the mTOR receptors, blocking DNA synthesis and the proliferation of T and B cells. The side effect profiles for sirolimus include myelosuppression, diarrhea, mouth ulcers, hyperlipidemia, refractory edema, and, most importantly, impaired wound healing [ 34 ].

The appearance of optical activity (chirality) has long been associated with molecular asymmetry. There are several types of chirality, but in natural organisms, homochirality (chiral purity) predominates—this is a type of chirality in which compounds are represented as a single isomer of two possible ones and remain unchanged in fundamental processes, as the absolute stereochemical configurations of L-amino acids [ 42 ]. As shown in the previous section, all generations of immunosuppressants, in addition to their direct action, have many side effects, often exceeding the benefit–harm balance. The only peptide immunosuppressant, Cyclosporine, has been used for many years and is still used to treat various autoimmune diseases and to suppress graft-versus-host disease (GVHD) treatment reactions and organ transplant rejection [ 26 ]. The minimal toxicity of endogenous peptides is advantageous over other classes of potential immunosuppressants. In our studies, the natural peptide Glu-Trp exhibited immunostimulating activity and is approved for medical use as Thymogen [ 43 ]. In structural and functional studies, we discovered that the enantiomeric dipeptide D-Glu-D-Trp and its analog D-Glu(D-Trp) exhibit immunosuppressive properties. Further research showed that these reciprocal reactions are based on the fundamental properties of the chirality of biological compounds [ 44 ]. Almost all chiral molecules in living organisms occur in only one form: sugars are represented by D-isomers, proteins composed of L-isomers, and DNA twists into right-handed helices. The biological regulation of homeostasis relies on homochiral molecules, such as amino acids, sugars, and DNA, to function correctly. Some researchers believe that this homochirality was a prerequisite for the formation of replicating molecules that gave rise to all life [ 45 , 46 ]. Most drug molecules have unique spatial structures, stereospecificity, and pharmacological activity. Therefore, determining the basic structural properties of the interaction between the peptide and its target is crucial to successful modifications to, and increasing the stability and maintaining the biological activity of, the potential drug candidate. The application of chirality in synthetic small-molecule drugs has been developed for a long time; the enantiomeric isomers may have the same physical and chemical properties, but depending on the stereoselectivity, the role of the enantiomers in the chiral environment may be very different. There is high interest in exploring peptide chirality for conjugating with existing non-peptide drugs, biopolymers, and nanomaterials to enhance their biomolecular interactions and selectivity. Though D-amino acids contain peptides and are present in trace amounts, the recent advances in analytical techniques permit more accurate analysis. Subsequently, this will assist in better understanding their role in disease development and progress [ 47 ]. Chiral dipeptides have gained attention as effective ligands in chiral therapeutics due to their biocompatibility, small size, and affordable functionalization potential [ 48 , 49 , 50 ]. The short stability and low toxicity of D-peptide isomers make them ideal for use in biomedicine for various applications, such as cancer treatment, vaccination, and neuronal differentiation [ 51 , 52 ]. Short oligopeptides and those of different sequences and lengths can be selectively engineered into specific compounds, such as self-assembling helical structures [ 53 ]. The homochiral optical purity is crucial for precise mechanism studies, obtaining regulatory approval, ensuring product consistency, and achieving pure chirality in the preparations [ 54 ]. While peptides derived from ribosomal synthesis are translated exclusively using L-amino acids, a formation of D-amino acid residues in animal peptides appears as a result of post-translational modification of an L-amino acid residue enzymatically converted into a D-amino acid residue in the peptide chain. The amount of free D-amino acids in the body is linked with many diseases. D-amino and D-amino acids contain peptides in some disease conditions, making them potential biomarkers and therapeutic targets for those diseases [ 55 ]. The serious challenge in pharmaceutical and biopharmaceutical chemistry is the preparation of effective drugs free of admixtures of side compounds, including unnatural optical isomers. Since the mechanisms of their action usually involve protein, carbohydrate, or nucleotide receptors that are also chiral, the observed activity is strictly stereospecific: only one of the two enantiomers efficiently interacts with the receptor. The second isomer is less active or not active at all. Unfortunately, there are examples when one (unnatural) enantiomer interacts with different receptors and induces dangerous biological effects. In some cases, as in a Thalidomide “story,” the racemic compound had an enantiomer that caused teratogenic deadly con

A novel situation arose with the discovery of the influence of Thymodepressisn on immune and hematopoietic systems. Non-post-translational modification for chemically prepared D-isomers of Glu-Trp peptides for the first time made it possible to use both enantiomers for up-and-down homeostasis regulation at the hemo- and immunopoiesis levels [ 3 ]. Short peptides’ low toxicity and stability make them ideal for use in chiral nanomedicine for various potential treatments, including cancer, neuronal differentiation, and vaccination [ 80 ]. The study of the nature of Thymodepressin’s suppressive effects on immune and hematopoietic systems required various in vitro and in vivo tests to understand its potential for clinical practice. Starting with the preclinical studies on Thymodepressin, H 3 -Thymodepressin distribution in animals was assessed in mice after a single intramuscular injection ( Table 1 ). A comparative analysis of the values of the areas under the pharmacokinetic curves (AUC) showed the extraordinary tropism of H 3 -Thymodepressin to the bone marrow since the AUC value for the bone marrow exceeded that for the blood by 22.6 times [ 81 ]. Unlike Thymogen, which targets CD4 and CD8 receptors on T lymphocytes, the binding of Thymodepressin to bone marrow cells indicates that its target is receptors located on bone marrow cells. In the fundamental experiments, the hemosuppressive activity of Thymodepressin on the proliferation of the hemopoietic precursors was evaluated in vitro in methylcellulose, using the mitochondria toxicity (MTT) test. The result indicates that Thymodepressin suppresses the cloning efficiency of all hematopoietic stem cell progenitors in a wide dose range (from 1 µg/mL to 10 µg/mL) [ 82 ]. In a systemic Thymodepressin study on bone marrow hematopoietic stem cells (HSCs), the only cells in the hematopoietic system that differentiated into all functional blood cell lineages, we used a revolutionary spleen colony-forming unit (CFU-S) assay developed for the assessment of the functional capacity of bone-marrow-derived hematopoietic progenitors at the single-cell level. This assay revealed the self-renewal and clonal differentiation capacity of hematopoietic progenitors through the transplantation of bone marrow cells and is still used in experimental research and clinical practice [ 83 ]. Experiments were carried out in a wide range of Thymodepressin conditions with various combination doses. One study determined the clonal differentiation capacity of many hematopoietic progenitors responsible for producing most CFU-S-8 splenic colonies using this assay [ 84 ]. Thymodepressin, in vitro and in vivo, affects the initial stages of hemopoiesis, reducing the number of committed CFU-C-8 cells in the S-phase of the cell cycle. As a result, administration of the peptide leads to a dose-dependent transient decrease in the number of leucocytes in the blood of the experimental animal [ 85 ]. Another level of Thymodepressin action on hematopoiesis is the suppression of mature T-lymphocyte activation, indicated by changed CD25+ and CD69+ markers [ 86 ]. Depending on the administration time, the unique Thymodepressin dual-direction action on activated cell clones can only be used in preventive or suppressive treatment. This new phenomenon was demonstrated in graft-versus-host disease (GVHD) treatment. When the thymodepressin-suppressing activity was discovered, the only peptide immunosuppressor, Cyclosporine, was approved for clinical practice [ 87 ]. Various in vitro and in vivo tests and preclinical and clinical studies have been performed to prove Thymodepressin’s suppressive effects on immune- and hematopoietic systems; some experiments were in direct comparison with Cyclosporin. To elucidate Tymodepressin’s and Cyclosporine’s influence on the colony-forming unit (CFU), the post-transplant effect on GVHD development was assessed by an allogeneic bone marrow test [ 88 ]. The test was based on the induction of chronic GVHD in hybrid non-irradiated mice with 100 million parent spleen cells, provoking the chronic GVHD. In this model, direct GVHD prevention by Tymodepressin and Cyclosporin was assessed. Thymodepressin was i/p-administered to lethally irradiated recipient mice (CBA × C57B1/6)F1 three days (1, 2, and 3 days) after allogeneic bone marrow transplantation [ 89 ]. Cyclosporin A was administered per/os in an oil solution at 0.5 mg in 0.2 mL/mouse thrice over three days. The optimal three-fold Thymodepressin administration increased the yield of colonies by approximately four times; Cyclosporin increased it only two times compared to the control animals [ 90 ]. Understanding the necessity of having a hematopoietic protector in medical practitioners’ arsenal from damaging factors such as radiation or cytostatics, we studied Thymodepressin as the potential preparation for these applications [ 91 ]. For Thymodepressin testing as a hematopoietic suppressor, the source of the bone marrow cells

The evolutionary predetermination of the homochirality in living organisms may give a chance to use the D-enantiomeric dipeptide antipode to gently slow down or block the developed negative process in the body. Our study of short immunotropic peptides showed the perspective of a broader study of various types of biological activities. While there are unsolved challenges, D-di-polypeptide research has a strong potential to generate an explosive impact on numerous research topics. The first milestone will be the complete assembly of an enantiomeric ribosome comprising D-proteins and L-rRNAs since only 3 out of 50 mirror-image E. coli ribosomal proteins and efficient L-nucleotide polymerases have been prepared. Our findings on such changes in the optical configuration of Glu-Trp isomers resulted in reciprocal changes in the magnitude and direction of their biological effects [ 3 , 66 , 67 , 70 , 71 , 77 ]. A significant difference between Tymodepressin and other drugs is its effect on activated cell clones without suppressing the activity of memory cells, i.e., without affecting previously acquired immunity. Thymodepressin reversibly affects any chronic autoimmune processes associated with sensitization to self-antigens and the production of autoantibodies. Positive results have also been obtained in treating the secondary immune cytopenia that develops against the background of lymphatic and other tumors. Thymodepressin’s hemoregulatory effect is directed at minimizing the consequences of the myelotoxic impacts of antitumor drugs due to a temporary delay in the differentiation of stem cells, providing a protective effect during the course of cytostatic therapy and a rapid and complete restoration of the granulocyte lineage. The effect is not canceled or reduced when combined with a cytostatic and TD. The Thymodepressin-positive antitumor effect on pathological cells can be extended to any antitumor course of therapy for various tumors. From an organic chemistry point of view, dipeptides are not only a minimal peptide sequence consisting of two different amino acids but an integral organic molecule whose general chemical structure and optical and spatial orientation determine the nature and the magnitude of its biological effects. Based on our experimental data, the dipeptide molecules are a unique “bridge” between polypeptides and the active substances of most modern synthetic drugs, such as proteins and low-molecular organic compounds (small molecules). The evolutionary predetermination of the homochirality in living organisms gives a chance to overcome this fundamental “restriction” and use the synthetic D-enantiomeric dipeptide antipodes to gently slow down or block the developed negative process in the body. The established relationships between the optical and chemical structures of the Glu-Trp dipeptides and their biological properties will help search for new peptide drug development areas. Understanding the possible applicability of this unusual reciprocal phenomenon to other endogenous dipeptides, other chiral polypeptides, and self-assembled suprapolimeric molecules opens additional options for detecting a reciprocal effect on their enantiomers. In the case that a general ability for the reciprocal regulation of organism homeostasis is widely proved, prophylactic and gentle up-and-down internal correction may occur without xenobiotics, cytostatics, and expensive monoclonal antibody (MAB) pharmaceuticals.