Additional file 1 of Discovery of a novel and highly selective JAK3 inhibitor as a potent hair growth promoter
Creators: name: Hossain, Md Mehedi
name: Khalid, Arfan
name: Akhter, Zaheen
name: Parveen, Sabra
name: Ayaz, Mir Owais
name: Bhat, Aadil Qadir
name: Badesra, Neetu
name: Showket, Farheen
name: Dar, Mohmmad Saleem
name: Ahmed, Farhan
name: Dhiman, Sumit
name: Kumar, Mukesh
name: Singh, Umed
name: Hussain, Razak
name: Keshari, Pankaj
name: Mustafa, Ghulam
name: Nargorta, Amit
name: Taneja, Neha
name: Gupta, Somesh
name: Mir, Riyaz A.
name: Kshatri, Aravind Singh
name: Nandi, Utpal
name: Khan, Nooruddin
name: Ramajayan, P.
name: Yadav, Govind
name: Ahmed, Zabeer
name: Singh, Parvinder Pal
name: Dar, Mohd Jamal
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. A (a) IC50 of MJ04 Km (ATP concentration). (b) IC50 of MJ04 at 1mM ATP concentration and (c) IC50 of Tofacitinib at Km ATP concentration. B–F RMSDs, RMSFs, and intermolecular interactions of MJ04 with JAK1, JAK2, and JAK3: B. Root Mean Square deviations (RMSDs). C. root mean Square Fluctuations (RMSFs) illustrating dynamic fluctuations during simulation. D, E, and F represent intermolecular interactions of MJ04 with JAK1, JAK2, and JAK3, respectively. S1. Table 1. Inhibition activities (IC50, nM) of small molecules against JAK3 kinase (Cell-free assay). S1. Table 2. Binding affinity and H-bonding interactions of ligand molecule (MJ04) in the hinge region of the kinase domain of JAK1, JAK2, and JAK3 proteins. S2 Table 1. Inhibition activities (IC50, μM) of small molecules against A549, HCT-116, Mia PaCa-2, MCF-7-2and Panc-2. Figure S2A. Schematic representation flow regarding selection of compounds against the target. S2B. General scheme for the synthesis of MJ04. S2C. 1H—NMR of MJ04. S2D. 19F—NMR of MJ04.S2E. HRMS of MJ04. Figure S3. Hair regrowth induced by MJ04 in a DHT-Induced Androgenetic Alopecia (AGA) mouse model. The experiment involved the daily treatment of the shaved dorsal skin of C57BL/6J mice with 0.5% testosterone for 1 h before the topical application of different concentrations of tofacitinib, MJ04, and baricitinib for 28 days. Digital photographs were taken from the representative area using a Nikon digital camera (n = 8 mice). (i)- Control group, (ii)-Testosterone group, (iii)-Vehicle group, (iv)- Tofacitinib group (0.8mg/Kg), (v)- Tofacitinib group (0.08 mg/kg), (vi)- MJ04 group (0.08 mg/kg), (vii) MJ04 group (0.04 mg/kg), (viii) MJ04 group (0.016 mg/kg), (ix) Baricitinib group (0.1 mg/kg), (x) Baricitinib group (0.04 mg/kg), and (xi) Baricitinib group (0.02 mg/kg). Figure S3L. Stability analysis of the formulation used for in vivo studies. The stability study of the lead compound (MJ04) was performed using HPLC analysis wherein the samples were studied after different time intervals. The 0.6 mg of MJ04 was taken and dissolved in 1 mL of ethanol to achieve 0.6 mg/mL concentration. Out of this, 500 μL was further diluted to 0.3 mg/mL by adding 500 μL ethanol to it (Ethanol sample). To remaining 500 μL, we added 500 μL PEG- 300 to achieve final concentration of 0.3 mg /mL (Ethanol: PEG sample). HP LC of these samples was performed at various time points (0 h, 12 h, 24h and 48 h) upon incubating at room temperature. The HPLC of samples was performed by RP-18 end capped; 5 μM, 4.6 x 250 mm. The mobile phase containing water (30%) and acetonitrile (70%) was used at the flow rate of 1 mL/min at a column temperature of 27 °C at 210 nm wavelength. A volume of 5 μL of sample was injected and the total run time of the assay was 30 min using the isocratic technique in the HPLC system. HPLC chromatograms are depicted in Figure A and Figure B. S3-Table 1. Weight of C57BL/6J mice. S3-Table 2. Biochemical Parameters in C57/B6 mice. S3-Table 3. Haematological Parameter in C57/B6 mice. Figure S4. MJ04 induces early onset in anagen development and hair cycle progression in nude mice (NU/J Foxn1nu). (A) MJ04 induced telogen-anagen transition, as shown by the dorsal skin turning from hairless to white hair. Digital photographs were taken from the representative area using a Nikon digital camera (n = 5 mice). (i)- Control group, (ii)-Vehicle group, (iii)- Tofacitinib group (0.8 mg/Kg), (iv)- Tofacitinib group (0.08 mg/kg), (v)- MJ04 group (0.08 mg/kg), (vi) MJ04 group (0.04 mg/kg), (vii) MJ04 group (0.016 mg/kg), (viii) Baricitinib group (0.1 mg/kg), (ix) Baricitinib group (0.04 mg/kg), and (x) Baricitinib group (0.02 mg/kg). Figure S5. Evaluation of toxicity of MJ04 and Tofacitinib in splenocytes by MTT assay. Figure S6. A Histological images of organs from subacute toxicity experiment. Organs stained by H&amp;E was obtained from the heart, liver, lung, and brain of Wistar rats after treatment with or without MJ04 (2000 mg/Kg). B Pharmacokinetic profile of MJ04, illustrating its dynamic behavior and disposition in C57BL/6 mice over a defined period of time.